Methods and compositions for use of therapeutic t cells in combination with kinase inhibitors

ABSTRACT

The present disclosure relates to methods, compositions and uses involving immunotherapies and inhibitors of a target protein tyrosine kinase in which the kinase is not IL-2-inducible T cell kinase (ITK) and/or is selected from Bruton&#39;s tyrosine kinase (BTK), tec protein tyrosine kinase (TEC), BMX non-receptor tyrosine kinase (Etk), TXK tyrosine kinase (TXK) and/or receptor tyrosine-protein kinase ErbB4 (ErbB4). The provided methods, compositions and uses include administration of one or more such inhibitor with another agent, such as an immunotherapeutic agent targeting T cells and/or genetically engineered T cells, such as CAR-expressing T cells. Also provided are methods of manufacturing engineered cells, cells, compositions, methods of administration, nucleic acids, articles of manufacture and kits. In some aspects, features of the methods and cells provide for improved activity, efficacy, persistence, expansion and/or proliferation of T cells for adoptive cell therapy or endogenous T cells recruited by immunotherapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/465,542 filed May 30, 2019 which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No. PCT/US2017/064362filed Dec. 1, 2017, which claims priority from U.S. provisionalapplication No. 62/429,732 filed Dec. 3, 2016, entitled “Methods andCompositions for Use of Therapeutic T cells in Combination with KinaseInhibitors” and to U.S. provisional application No. 62/581,644 filedNov. 3, 2017, entitled “Methods and Compositions for Use of TherapeuticT cells in Combination with Kinase Inhibitors,” the contents of whichare incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the electronic Sequence Listing (file name:735042008901SeqList.xml, date created: Dec. 16, 2022, size: 29,570bytes) is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates in some aspects to methods, compositionsand uses involving immunotherapies, such as adoptive cell therapy, e.g.,T cell therapy, in combination or conjunction with modulators, e.g.,inhibitors, of a target kinase, such as a target protein tyrosinekinase. Also provided are methods of manufacturing such engineeredcells, cells, compositions, such as methods in which the cells areproduced in the presence of one or more of the kinase inhibitors. Alsoprovided are methods of administration to subjects, nucleic acids,articles of manufacture and kits for use in the methods.

BACKGROUND

Various strategies are available for immunotherapy, for example,adoptive cell therapy methods involving administering T cells, such asgenetically engineered antigen receptors, such as CARs. In some aspects,available methods may not be entirely satisfactory. There is a need foradditional strategies for immunotherapy and adoptive cell therapy, e.g.,strategies to enhance persistence, activity and/or proliferation ofadministered cells and responses and strategies for modulating T cellphenotype. Provided are methods, cells, compositions, kits, and systemsthat meet such needs.

SUMMARY

Provided herein are methods of enhancing or modulating proliferationand/or activity of T cell activity associated with administration of animmunotherapy or immunotherapeutic agent, such as a compositionincluding cells for adoptive cell therapy, e.g., such as a T celltherapy (e.g. CAR-expressing T cells) or a T cell-engaging therapeuticagent, such as a bispecific or multispecific agent or antibody, capableof recruiting one or more T cells or other immune cells. In someaspects, such methods included combination administration of theimmunotherapy or immunotherapeutic agent and an inhibitor of one or morenon-receptor protein tyrosine kinases such as TEC family kinases. Insome aspects, the protein tyrosine kinase is not IL-2-inducible T cellkinase (not an ITK) and/or the inhibitor does not inhibit ITK, e.g.,does not inhibit ITK with an IC50 value less than 1000 or less than 500nM. In some aspects, the target protein tyrosine kinase is a Bruton'styrosine kinase (BTK), a tec protein tyrosine kinase (TEC), a BMXnon-receptor tyrosine kinase (Etk), a TXK tyrosine kinase (TXK) and/or areceptor tyrosine-protein kinase ErbB4 (ErbB4). The provided methods,compositions and uses include those for combination therapies involvingthe administration or use of one or more such inhibitor in conjunctionwith another agent, such as an immunotherapeutic agent that involves,recruits or engages T cells targeting a disease or condition, such as atherapeutic antibody, e.g., a multispecific (e.g., T cell engaging)antibody, and/or therapeutic compositions containing immune cells, suchas T cells, such as genetically engineered T cells, such as chimericantigen receptor (CAR)-expressing T cells. In some aspects, features ofthe methods and cells provide for increased or improved activity,efficacy, persistence, expansion and/or proliferation of T cells foradoptive cell therapy or endogenous T cells recruited byimmunotherapeutic agents.

In some embodiments, the methods generally involve administrating acombination therapy of the immunotherapy or immunotherapeutic agent,such as a composition including cells for adoptive cell therapy, e.g.,such as a T cell therapy (e.g. CAR-expressing T cells) or a Tcell-engaging therapeutic agent and an inhibitor of a target proteintyrosine kinase that is not IL-2-inducible T cell kinase (ITK) and/or inwhich the target protein tyrosine kinase is selected from Bruton'styrosine kinase (BTK), tec protein tyrosine kinase (TEC), BMXnon-receptor tyrosine kinase (Etk), TXK tyrosine kinase (TXK) and/orreceptor tyrosine-protein kinase ErbB4 (ErbB4).

Provided herein are methods of treatment that involve: (a)administering, to a subject having a disease or condition, T cells thatspecifically recognize or specifically bind to an antigen associatedwith, or expressed or present on cells of, the disease or conditionand/or a tag comprised by a therapeutic agent that specifically targetsthe disease or condition and has been or is to be administered to thesubject; and (b) administering to the subject an inhibitor of a targetprotein tyrosine kinase, wherein the inhibitor does not inhibit ITKand/or inhibits ITK with a half-maximal inhibitory concentration (IC50)of greater than or greater than about 1000 nM, wherein the disease orcondition (i) is not a B cell-derived disease or condition (ii) is notassociated with expression of CD19, CD22, or CD20; (iii) does notexpress the target protein tyrosine kinase, (iv) does not contain a formof the target protein tyrosine kinase that is sensitive to theinhibitor, (v) does not contain a kinase sensitive to the inhibitorand/or (vi) is not sensitive to inhibition by the inhibitor and/orwherein the subject or disease or condition is resistant or refractoryto the inhibitor and/or to an inhibitor of BTK and/or wherein theprotein tyrosine kinase is not ordinarily expressed or is not suspectedof being expressed in cells from which the disease or condition isderived.

Provided in some aspects are methods of treatment that involveadministering, to a subject having a disease or condition, T cells thatspecifically recognize or specifically bind to an antigen associatedwith, or expressed or present on cells of, the disease or conditionand/or a tag comprised by a therapeutic agent that specifically targetsthe disease or condition and has been or is to be administered to thesubject, wherein: the subject has been administered an inhibitor of atarget protein tyrosine kinase, wherein the inhibitor does not inhibitITK and/or inhibits ITK with a half-maximal inhibitory concentration(IC50) of greater than or greater than about 1000 nM; and the disease orcondition (i) is not a B cell-derived disease or condition (ii) is notassociated with expression of CD19, CD22, or CD20; (iii) does notexpress the protein tyrosine kinase, (iv) does not contain a form of thetarget protein tyrosine kinase that is sensitive to the inhibitor, (v)does not contain a kinase sensitive to the inhibitor and/or (vi) is notsensitive to inhibition by the inhibitor and/or wherein the subject ordisease or condition is resistant or refractory to the inhibitor and/orto an inhibitor of BTK and/or wherein the protein tyrosine kinase is notordinarily expressed or is not suspected of being expressed in cellsfrom which the disease or condition is derived.

Provided herein are methods of treatment that involve administering, toa subject having a disease or condition, an inhibitor of a targetprotein tyrosine kinase, wherein the inhibitor does not inhibit ITKand/or inhibits ITK with a half-maximal inhibitory concentration (IC50)of greater than or greater than about 1000 nM, wherein: the subject hasbeen administered T cells that specifically recognize or specificallybind to an antigen associated with, or expressed or present on cells of,the disease or condition and/or a tag comprised by a therapeutic agentthat specifically targets the disease or condition and has been or is tobe administered to the subject; and the disease or condition (i) is nota B cell-derived disease or condition (ii) is not associated withexpression of CD19, CD22, or CD20; (iii) does not express the targetprotein tyrosine kinase, (iv) does not contain a form of the targetprotein tyrosine kinase that is sensitive to the inhibitor, (v) does notcontain a kinase sensitive to the inhibitor and/or (vi) is not sensitiveto inhibition by the inhibitor and/or wherein the subject or disease orcondition is resistant or refractory to the inhibitor and/or to aninhibitor of BTK and/or wherein the TEC family kinase is not ordinarilyexpressed or is not suspected of being expressed in cells from which thedisease or condition is derived.

In some embodiments of any of the methods provided herein, the targetprotein tyrosine kinase is tyrosine kinase expressed in hepatocellularcarcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a BMX/ETK, or anERBB4. In some embodiments, the target protein tyrosine kinase is a TECfamily kinase.

In some embodiments of any of the methods provided herein, the inhibitoris selected from Formula (II), ONO/GS-4059, Compound 30 or Compound 38,GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;CNX-774; and LFM-A13.

In some embodiments of any of the methods provided herein, the inhibitoris a selective inhibitor of the target protein tyrosine kinase. In someembodiments, the inhibitor inhibits the target protein tyrosine kinasewith a half-maximal inhibitory concentration (IC50) that is at least 10or at least 100 times lower than that of the IC50 of the inhibitor forany additional protein tyrosine kinase or TEC family kinase, and/orinhibits the target protein tyrosine kinase with an IC50 at least 2, atleast 10 or at least 100 times lower than that the IC50 value of theinhibitor for both ITK and BTK. In some embodiments, the inhibitorinhibits the target protein tyrosine kinase with a half-maximalinhibitory concentration (IC50) of less than or less than about 1000 nM,900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM or less

Provided herein are methods that involve: (1) administering, to asubject having a disease or condition, T cells that specificallyrecognize or specifically bind to an antigen associated with the diseaseor condition and/or a tag comprised by a therapeutic agent thatspecifically targets the disease or condition and has been or is to beadministered to the subject; and (2) administering to the subject aninhibitor of a target protein tyrosine kinase, which target proteintyrosine kinase is a tyrosine kinase expressed in hepatocellularcarcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a BMX/ETK, or anERBB4. In some embodiments, the inhibitor is a selective inhibitor ofthe target protein tyrosine kinase. In some embodiments, the inhibitorinhibits the target protein tyrosine kinase with a half-maximalinhibitory concentration (IC50) that is at least 10 or at least 100times lower than that of the IC50 of the inhibitor for any proteintyrosine kinase or TEC family kinase distinct from the target proteintyrosine kinase, and/or inhibits the target protein tyrosine kinase withan IC50 at least 2, at least 10 or at least 100 times lower than thatthe IC50 value of the inhibitor for both ITK and BTK. In someembodiments, the inhibitor inhibits the target protein tyrosine kinasewith a half-maximal inhibitory concentration (IC50) of less than or lessthan about 1000 nM, 900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200nM, 100 nM or less.

Provided herein are methods that involve administering, to a subjecthaving a disease or condition, T cells that specifically recognize orspecifically bind to an antigen associated with the disease or conditionand/or a tag comprised by a therapeutic agent that specifically targetsthe disease or condition and has been or is to be administered to thesubject, said subject having been administered an inhibitor of a targetprotein tyrosine kinase, which target protein tyrosine kinase is atyrosine kinase expressed in hepatocellular carcinoma (TEC), a restinglymphocyte kinase (RLK/TXK), a BMX/ETK, or an ERBB4. In someembodiments, the inhibitor is a selective inhibitor of the targetprotein tyrosine kinase. In some embodiments of any of the methodsprovided herein, the inhibitor inhibits the target protein tyrosinekinase with a half-maximal inhibitory concentration (IC50) that is atleast 10 or at least 100 times lower than that of the IC50 of theinhibitor for any protein tyrosine kinase or TEC family kinase distinctfrom the target protein tyrosine kinase, and/or inhibits the targetprotein tyrosine kinase with an IC50 at least 2, at least 10 or at least100 times lower than that the IC50 value of the inhibitor for both ITKand BTK. In some embodiments of any of the methods provided herein, theinhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) of less than or less thanabout 1000 nM, 900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM,100 nM or less.

Provided herein are methods of treatment that involve administering to asubject, having a disease or condition, an inhibitor of a target proteintyrosine kinase, which target protein tyrosine kinase is a tyrosinekinase expressed in hepatocellular carcinoma (TEC), a resting lymphocytekinase (RLK/TXK), a BMX/ETK, or an ERBB4, said subject having beenadministered T cells that specifically recognize or specifically bind toan antigen associated with the disease or condition and/or a tagcomprised by a therapeutic agent that specifically targets the diseaseor condition and has been or is to be administered to the subject. Insome embodiments, the inhibitor is a selective inhibitor of the targetprotein tyrosine kinase. In some embodiments of any of the methodsprovided herein, the inhibitor inhibits the target protein tyrosinekinase with a half-maximal inhibitory concentration (IC50) that is atleast 10 or at least 100 times lower than that of the IC50 of theinhibitor for any protein tyrosine kinase or TEC family kinase distinctfrom the target protein tyrosine kinase, and/or inhibits the targetprotein tyrosine kinase with an IC50 at least 2, at least 10 or at least100 times lower than that the IC50 value of the inhibitor for both ITKand BTK. In some embodiments of any of the methods provided herein, theinhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) of less than or less thanabout 1000 nM, 900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM,100 nM or less.

In some embodiments of any of the methods provided herein, the diseaseor condition (i) is not a B cell-derived disease or condition (ii) isnot associated with expression of CD19, CD22, or CD20; (iii) does notexpress the target protein tyrosine kinase, (iv) does not contain a formof the target protein tyrosine kinase that is sensitive to theinhibitor, (v) does not contain a kinase sensitive to the inhibitorand/or (vi) is not sensitive to inhibition by the inhibitor and/orwherein the subject or disease or condition is resistant or refractoryto the inhibitor and/or to an inhibitor of BTK and/or the target kinaseis not ordinarily expressed or is not suspected of being expressed incells from which the disease or condition is derived. In someembodiments of any of the methods provided herein, the target proteintyrosine kinase is a RLK/TXK.

In some embodiments of any of the methods provided herein, the targetprotein tyrosine kinase is a BMX/ETK and the inhibitor inhibits Bmx/Etkwith an a half-maximal inhibitory concentration (IC50) that is at least10 or at least 100 times lower than that of the IC50 of the inhibitorfor any other TEC family kinase and/or for ITK, and/or inhibits Bmx/Etkwith a half-maximal inhibitory concentration (IC50) of less than or lessthan about 1000 nM, 900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200nM, 100 nM or less. In some embodiments of any of the methods providedherein, the target kinase is or contains an ErbB4.

In some embodiments of any of the methods provided herein, the inhibitorcontains is a compound of formula (II):

(II), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof.

In some embodiments of any of the methods provided herein, the inhibitorcontains the compound of Formula (II), or an enantiomer,pharmaceutically-acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof or a pharmaceutical composition comprisingany of the foregoing.

Provided in some aspects are methods of treatment that involve (1)administering, to a subject having a disease or condition, T cellscomprising a recombinant antigen receptor that specifically binds to anantigen associated with the disease or condition and/or a tag comprisedby a therapeutic agent that specifically targets the disease orcondition and has been or is to be administered to the subject; and (2)administering to the subject a kinase inhibitor or a pharmaceuticalcomposition comprising the inhibitor, wherein the inhibitor contains thecompound of Formula (II), or an enantiomer, pharmaceutically-acceptablesalt, solvate, hydrate, co-crystal, polymorph or prodrug thereof.

Provided in some aspects are methods of treatment that involveadministering, to a subject having a disease or condition, T cellscomprising a recombinant antigen receptor that specifically binds to anantigen associated with the disease or condition and/or a tag comprisedby a therapeutic agent that specifically targets the disease orcondition and has been or is to be administered to the subject, saidsubject having been administered a kinase inhibitor or a pharmaceuticalcomposition comprising the inhibitor, wherein the inhibitor contains thecompound of Formula (II) or an enantiomer, pharmaceutically-acceptablesalt, solvate, hydrate, co-crystal, polymorph or prodrug thereof.

Provided in some aspects are methods of treatment that involveadministering, to a subject having a disease or condition, a kinaseinhibitor or a pharmaceutical composition comprising the inhibitor,wherein the inhibitor contains the compound of Formula (II), or anenantiomer, pharmaceutically-acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof, said subject having beenadministered T cells comprising a recombinant antigen receptor thatspecifically binds to an antigen associated with the disease orcondition and/or a tag comprised by a therapeutic agent thatspecifically targets the disease or condition and has been or is to beadministered to the subject.

In some embodiments of any of the methods provided herein, the diseaseor condition is a cancer.

In some embodiments of any of the methods provided herein, (i) thesubject and/or the disease or condition (a) is resistant to inhibitionof Bruton's tyrosine kinase (BTK) and/or (b) comprises a population ofcells that are resistant to inhibition by the inhibitor; (ii) thesubject and/or the disease or condition contains a mutation ordisruption in a nucleic acid encoding BTK, capable of reducing orpreventing inhibition of the BTK by the inhibitor and/or by ibrutinib;and/or (iii) at the time of the administration in (1) and at the time ofthe administration in (2) the subject has relapsed following remissionafter treatment with, or been deemed refractory to treatment with theinhibitor and/or with a BTK inhibitor therapy. In some embodiments, thepopulation of cells is or comprises a population of B cells and/or doesnot include T cells. In some embodiments, the mutation in the nucleicacid encoding BTK contains a substitution at position C481, optionallyC481S or C481R, and/or a substitution at position T474, optionally T474Ior T474M.

In some embodiments of any of the methods provided herein, the targetprotein tyrosine kinase is not expressed by cells of the disease orcondition, is not ordinarily expressed or is not suspected of beingexpressed in cells from which the disease or condition is derived,and/or the disease or condition is not sensitive to the inhibitor;and/or at least a plurality of the T cells express the target proteintyrosine kinase; and/or the target protein tyrosine kinase is expressedin T cells.

In some embodiments of any of the methods provided herein, the diseaseor condition is a cancer not expressing a B cell antigen, anon-hematologic cancer, is not a B cell malignancy, is not a B cellleukemia, or is a solid tumor.

In some embodiments of any of the methods provided herein, the diseaseor condition is a cancer selected from sarcomas, carcinomas, lymphomas,non-Hodgkin lymphomas (NHLs), diffuse large B cell lymphoma (DLBCL),leukemia, CLL, ALL, AML and myeloma.

In some embodiments of any of the methods provided herein, the diseaseor condition is a pancreatic cancer, bladder cancer, colorectal cancer,breast cancer, prostate cancer, renal cancer, hepatocellular cancer,lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectalcancer, thyroid cancer, uterine cancer, gastric cancer, esophagealcancer, head and neck cancer, melanoma, neuroendocrine cancers, CNScancers, brain tumors, bone cancer, or soft tissue sarcoma.

In some embodiments of any of the methods provided herein, the T cellsrecognize or target an antigen selected from ROR1, B cell maturationantigen (BCMA), tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA,and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30,CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbBdimers, EGFR vIII, FBP, FCRLS, FCRHS, fetal acethycholine e receptor,GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain,Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associatedantigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen ofmelanoma (PRAIVIE), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptora2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1,HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands,CD44v6, dual antigen, and an antigen associated with a universal tag, acancer-testes antigen, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), prostate specific antigen, PSMA,Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, and a pathogen-specific antigen.

In some embodiments of any of the methods provided herein, the antigenis not a B cell antigen; and/or the antigen is not a B cell antigenselected from CD19, CD20, CD22, and ROR1. In some embodiments, theantigen is not a B cell antigen selected from CD19, CD20, CD22, andROR1; and/or the disease or condition does not express a B cell antigenselected from CD19, CD20, CD22 and ROR1 and/or kappa light chain.

In some embodiments of any of the methods provided herein, the T cellscontains tumor infiltrating lymphocytes (TILs) or contains geneticallyengineered T cells expressing a recombinant receptor that specificallybinds to the antigen. In some embodiments, the T cells comprisegenetically engineered T cells expressing a recombinant receptor thatspecifically binds to the antigen or the tag, which receptor optionallyis a chimeric antigen receptor. In some embodiments, the recombinantreceptor is a transgenic T cell receptor (TCR) or a functional non-Tcell receptor. In some embodiments, the recombinant receptor is achimeric receptor, which optionally is a chimeric antigen receptor(CAR). In some embodiments, the chimeric antigen receptor (CAR) includesan extracellular antigen-recognition domain that specifically binds tothe antigen and an intracellular signaling domain comprising an ITAM. Insome embodiments, the intracellular signaling domain includes anintracellular domain of a CD3-zeta (CD3) chain. In some embodiments, thechimeric antigen receptor (CAR) further contains a costimulatorysignaling region. In some embodiments, the costimulatory signalingregion contains a signaling domain of CD28 or 4-1BB. In someembodiments, the costimulatory domain is a domain of CD28.

In some embodiments of any of the methods provided herein, the inhibitoris a small molecule, peptide, protein, antibody or antigen-bindingfragment thereof, an antibody mimetic, an aptamer, or a nucleic acidmolecule.

In some embodiments of any of the methods provided herein, the inhibitorirreversibly reduces or eliminates the activation of the target proteintyrosine kinase, specifically binds to a binding site in the active siteof the target protein tyrosine kinase comprising an amino acid residuecorresponding to residue C481 in the sequence set forth in SEQ ID NO:18,and/or reduces or eliminates autophosphorylation activity of the targetprotein tyrosine kinase.

In some embodiments of any of the methods provided herein, the inhibitoris not ibrutinib. In some embodiments of any of the methods providedherein, the inhibitor is not the compound of Formula (II). In someembodiments of any of the methods provided herein, the inhibitor is notGDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;CNX-774; and LFM-A13.

In some embodiments of any of the methods provided herein, the inhibitoris administered concurrently with or subsequently to initiation ofadministration of the composition comprising the T cells.

In some embodiments of any of the methods provided herein, the inhibitoris administered subsequently to initiation of administration of the Tcells.

In some embodiments of any of the methods provided herein, the inhibitoris administered within, or within about, 1 hour, 2 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours or 1 week of theinitiation of the administration of the T cells.

In some embodiments of any of the methods provided herein, the inhibitoris administered at a time in which: the number of administered T cellsdetectable in the blood from the subject is decreased compared to in thesubject at a preceding time point after initiation of the administrationof the T cells; the number of administered T cells detectable in theblood is less than or less than about 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 50-fold or 100-fold or less the peak or maximum numberof the cells of the T cell therapy detectable in the blood of thesubject after initiation of administration of the administration of theT cells; and/or at a time after a peak or maximum level of theadministered T cells are detectable in the blood of the subject, thenumber of cells of or derived from the T cells detectable in the bloodfrom the subject is less than less than 10%, less than 5%, less than 1%or less than 0.1% of total peripheral blood mononuclear cells (PBMCs) inthe blood of the subject.

In some embodiments, the increase or decrease is by greater than orgreater than about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,10-fold or more.

In some embodiments of any of the methods provided herein, the inhibitoris administered for a time period up to 2 days, up to 7 days, up to 14days, up to 21 days, up to one month, up to two months, up to threemonths, up to 6 months or up to 1 year after initiation of theadministration of the administration of the T cells.

In some embodiments of any of the methods provided herein, the inhibitoris administered up to 3 months after initiation of the administration ofthe T cells.

In some embodiments of any of the methods provided herein, theadministration of the inhibitor is continued, from at least afterinitiation of administration of the T cells, until: the number of cellsof or derived from the T cells administered detectable in the blood fromthe subject is increased compared to in the subject at a preceding timepoint just prior to administration of the inhibitor or compared to apreceding time point after administration of the T-cell therapy; thenumber of cells of or derived from the T cells detectable in the bloodis within 2.0-fold (greater or less) the peak or maximum number observedin the blood of the subject after initiation of administration of the Tcells; the number of cells of the T cells detectable in the blood fromthe subject is greater than or greater than about 10%, 15%, 20%, 30%,40%, 50%, or 60% total peripheral blood mononuclear cells (PBMCs) in theblood of the subject; and/or the subject exhibits a reduction in tumorburden as compared to tumor burden at a time immediately prior to theadministration of the T cells or at a time immediately prior to theadministration of the inhibitor; and/or the subject exhibits complete orclinical remission.

In some embodiments of any of the methods provided herein, the inhibitoris administered orally, subcutaneously or intravenously. In someembodiments, the inhibitor is administered orally.

In some embodiments of any of the methods provided herein, the inhibitoris administered six times daily, five times daily, four times daily,three times daily, twice daily, once daily, every other day, three timesa week or at least once a week. In some embodiments of any of themethods provided herein, the inhibitor is administered once daily ortwice a day.

In some embodiments of any of the methods provided herein, the inhibitoris administered at a total daily dosage amount of at least or at leastabout 50 mg/day, 100 mg/day, 150 mg/day, 175 mg/day, 200 mg/day, 250mg/day, 300 mg/day, 350 mg/day, 400 mg/day, 450 mg/day, 500 mg/day, 600mg/day, 700 mg/day, 800 mg/day or more. In some embodiments, theinhibitor is administered in an amount less than or about less than orabout or 420 mg per day.

In some embodiments of any of the methods provided herein, theadministered T cells contain T cells that are CD4+ or CD8+. In someembodiments of any of the methods provided herein, the administered Tcells contain cells that are autologous to the subject. In someembodiments of any of the methods provided herein, the administered Tcells contain T cells that are allogeneic to the subject. In someembodiments of any of the methods provided herein, the administered Tcells include administration of a dose comprising a number of cellsbetween or between about 5×105 cells/kg body weight of the subject and1×107 cells/kg, 0.5×106 cells/kg and 5×106 cells/kg, between or betweenabout 0.5×106 cells/kg and 3×106 cells/kg, between or between about0.5×106 cells/kg and 2×106 cells/kg, between or between about 0.5×106cells/kg and 1×106 cell/kg, between or between about 1.0×106 cells/kgbody weight of the subject and 5×106 cells/kg, between or between about1.0×106 cells/kg and 3×106 cells/kg, between or between about 1.0×106cells/kg and 2×106 cells/kg, between or between about 2.0×106 cells/kgbody weight of the subject and 5×106 cells/kg, between or between about2.0×106 cells/kg and 3×106 cells/kg, or between or between about 3.0×106cells/kg body weight of the subject and 5×106 cells/kg, each inclusive.

In some embodiments of any of the methods provided herein, the dose ofcells administered is less than the dose in a method in which theadministered T cells are administered without administering theinhibitor. In some embodiments, the dose is at least 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold or 10-fold less.

In some embodiments of any of the methods provided herein, the T cellsare administered in a single dose, which optionally is a singlepharmaceutical composition comprising the cells.

In some embodiments of any of the methods provided herein, the T cellsare administered as a split dose, wherein the cells of a single dose areadministered in a plurality of compositions, collectively comprising thecells of the dose, over a period of no more than three days and/or themethod further includes administering one or more additional doses ofthe T cells.

In some embodiments of any of the methods provided herein, the methodfurther includes administering a lymphodepleting chemotherapy prior toadministration of the T cells and/or wherein the subject has received alymphodepleting chemotherapy prior to administration of the T cells.

In some embodiments of any of the methods provided herein, thelymphodepleting chemotherapy includes administering fludarabine and/orcyclophosphamide to the subject.

In some embodiments of any of the methods provided herein, the methodfurther includes: administering an immune modulatory agent to thesubject, wherein the administration of the cells and the administrationof the immune modulatory agent are carried out simultaneously,separately or in a single composition, or sequentially, in either order.In some embodiments, the immune modulatory agent is capable ofinhibiting or blocking a function of a molecule, or signaling pathwayinvolving said molecule, wherein the molecule is an immune-inhibitorymolecule and/or wherein the molecule is an immune checkpoint molecule.

In some embodiments, the immune checkpoint molecule or pathway isselected from PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine2A Receptor (A2AR), or adenosine or a pathway involving any of theforegoing.

In some embodiments of any of the methods provided herein, the immunemodulatory agent is or contains an antibody, which optionally is anantibody fragment, a single-chain antibody, a multispecific antibody, oran immunoconjugate.

In some embodiments, the antibody specifically binds to the immunecheckpoint molecule or a ligand or receptor thereof; and/or the antibodyis capable of blocking or impairing the interaction between the immunecheckpoint molecule and a ligand or receptor thereof.

In some embodiments of any of the methods provided herein, theadministered T cells exhibit increased or prolonged expansion and/orpersistence in the subject as compared to a method in which theadministered T cells are administered to the subject in the absence ofthe inhibitor.

In some embodiments of any of the methods provided herein, the methodreduces tumor burden to a greater degree and/or for a greater period oftime as compared to the reduction that would be observed with acomparable method in which the administered T cells are administered tothe subject in the absence of the inhibitor.

Provided in some aspects are combinations containing: geneticallyengineered T cells expressing a recombinant receptor that binds to anantigen other than a B cell antigen or other than a B cell antigenselected from CD19, CD20, CD22 and ROR1, and an inhibitor of a targetprotein tyrosine kinase, wherein the inhibitor does not inhibit ITKand/or inhibits ITK with a half-maximal inhibitory concentration (IC50)of greater than or greater than about 1000 nM and/or the target proteintyrosine kinase is a tyrosine kinase expressed in hepatocellularcarcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a BMX/ETK, or anERBB4.

In some embodiments of any of the combinations herein, the antigen isselected from among Her2, L1-CAM, mesothelin, CEA, hepatitis B surfaceantigen, anti-folate receptor, CD23, CD24, CD38, CD44, EGFR, EGP-2,EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR vIII, FBP, FCRLS, FCRHS,fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha,IL-13R-alpha2, kdr, Lewis Y, L1-cell adhesion molecule (L1-CAM),Melanoma-associated antigen (MAGEMAGE-A1, MAGE-A3, MAGE-A6,Preferentially expressed antigen of melanoma (PRAIVIE), survivin, EGP2,EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, GD3, HMW-MAA,CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folatereceptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors,5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, and an antigenassociated with a universal tag, a cancer-testes antigen, mesothelin,MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetalantigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostatespecific antigen, PSMA, estrogen receptor, progesterone receptor,ephrinB2, CD123, c-Met, GD-2 O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1(WT-1), a cyclin, cyclin A2, CCL-1, CD138, and a pathogen-specificantigen.

In some embodiments of any of the combinations herein, the antigen is apathogen-specific antigen, which is a viral antigen, bacterial antigenor parasitic antigen.

In some embodiments of any of the combinations herein, the recombinantreceptor is a transgenic T cell receptor (TCR) or a functional non-Tcell receptor.

In some embodiments of any of the combinations herein, the recombinantreceptor is a chimeric receptor, which optionally is a chimeric antigenreceptor (CAR).

In some embodiments of any of the combinations herein: the inhibitor isa selective inhibitor of the target protein tyrosine kinase; and/or theinhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) that is at least 10 or atleast 100 times lower than that of the IC50 of the inhibitor for anyprotein tyrosine kinase or TEC family kinase distinct from the targetprotein tyrosine kinase, and/or inhibits the target protein tyrosinekinase with an IC50 at least 2, at least 10 or at least 100 times lowerthan that the IC50 value of the inhibitor for both ITK and BTK; and/orthe inhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) of less than or less thanabout 1000 nM, 900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM,100 nM or less.

In some embodiments of any of the combinations herein, the inhibitor isa small molecule, peptide, protein, antibody or antigen-binding fragmentthereof, an antibody mimetic, an aptamer, or a nucleic acid molecule.

In some embodiments of any of the combinations herein, the inhibitor isselected from Formula (II), ONO/GS-4059, Compound 30 or Compound 38,GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;CNX-774; and LFM-A13.

In some embodiments of any of the combinations herein, the inhibitorcontains the compound of Formula (II), or an enantiomer,pharmaceutically-acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof or a pharmaceutical composition comprisingany of the foregoing.

In some embodiments of any of the combinations herein, the combinationis formulated in the same composition. In some embodiments of any of thecombinations herein, the combination is formulated in separatecompositions.

Provided herein are kits that contain the combination of any of claims76-86 and instructions for administering, to a subject for treating adisease or condition, optionally a cancer, the genetically engineeredcells and the inhibitor.

Provided herein are kits that contain a composition comprising atherapeutically effective amount of genetically engineered T cellsexpressing a recombinant receptor that binds to an antigen other than aB cell antigen or other than a B cell antigen selected from CD19, CD20,CD22 and ROR1; and instructions for administering, to a subject fortreating a cancer, the genetically engineered cells in a combinedtherapy with an inhibitor of a target protein tyrosine kinase, whereinthe inhibitor does not inhibit ITK and/or inhibits ITK with ahalf-maximal inhibitory concentration (IC50) of greater than or greaterthan about 1000 nM and/or the target protein tyrosine kinase is atyrosine kinase expressed in hepatocellular carcinoma (TEC), a restinglymphocyte kinase (RLK/TXK), a BMX/ETK, or an ERBB4.

Provided herein are kits that contain a composition comprising atherapeutically effective amount of an inhibitor of a target proteintyrosine kinase, wherein the inhibitor does not inhibit ITK and/orinhibits ITK with a half-maximal inhibitory concentration (IC50) ofgreater than or greater than about 1000 nM and/or the target proteintyrosine kinase is a tyrosine kinase expressed in hepatocellularcarcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a BMX/ETK, or anERBB4; and instructions for administering, to a subject for treating adisease or condition, optionally a cancer, the inhibitor in a combinedtherapy with genetically engineered T cells, said T cells expressing arecombinant receptor that binds to an antigen other than a B cellantigen or other than a B cell antigen selected from CD19, CD20, CD22and ROR1.

In some embodiments of any of the embodiments herein, the cancer is nota cancer expressing a B cell antigen, is a non-hematologic cancer, isnot a B cell malignancy, is not a B cell leukemia, or is a solid tumor.

In some embodiments of any of the embodiments herein, the cancer is asarcoma, a carcinoma or a lymphoma, optionally a non-Hodgkin lymphomas(NHLs), diffuse large B cell lymphoma (DLBCL), leukemia, CLL, ALL, AMLand myeloma.

In some embodiments of any of the embodiments herein, the cancer is apancreatic cancer, bladder cancer, colorectal cancer, breast cancer,prostate cancer, renal cancer, hepatocellular cancer, lung cancer,ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer,thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, headand neck cancer, melanoma, neuroendocrine cancers, CNS cancers, braintumors, bone cancer, or soft tissue sarcoma.

In some embodiments of any of the embodiments herein, (i) the subjectand/or the disease or condition (a) is resistant to inhibition ofBruton's tyrosine kinase (BTK) and/or (b) contains a population of cellsthat are resistant to inhibition by the inhibitor; (ii) the subjectand/or the disease or condition contains a mutation or disruption in anucleic acid encoding BTK, capable of reducing or preventing inhibitionof the BTK by the inhibitor and/or by ibrutinib; and/or (iii) at thetime of the administering the subject has relapsed following remissionafter treatment with, or been deemed refractory to treatment with theinhibitor and/or with a BTK inhibitor therapy.

In some embodiments of any of the embodiments herein, the population ofcells is or contains a population of B cells and/or does not contain Tcells.

In some embodiments of any of the embodiments herein, the mutation inthe nucleic acid encoding BTK contains a substitution at position C481,optionally C481S or C481R, and/or a substitution at position T474,optionally T474I or T474M.

In some embodiments of any of the embodiments herein, the antigen isselected from among Her2, L1-CAM, mesothelin, CEA, hepatitis B surfaceantigen, anti-folate receptor, CD23, CD24, CD38, CD44, EGFR, EGP-2,EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR vIII, FBP, FCRLS, FCRHS,fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha,IL-13R-alpha2, kdr, Lewis Y, L1-cell adhesion molecule (L1-CAM),Melanoma-associated antigen (MAGEMAGE-A1, MAGE-A3, MAGE-A6,Preferentially expressed antigen of melanoma (PRAIVIE), survivin, EGP2,EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, GD3, HMW-MAA,CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folatereceptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors,5T4, Fetal AchR, NKG2D ligands, CD44v6, dual antigen, and an antigenassociated with a universal tag, a cancer-testes antigen, mesothelin,MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetalantigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostatespecific antigen, PSMA, estrogen receptor, progesterone receptor,ephrinB2, CD123, c-Met, GD-2 O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1(WT-1), a cyclin, cyclin A2, CCL-1, CD138, and a pathogen-specificantigen.

In some embodiments of any of the embodiments herein, the antigen is apathogen-specific antigen, which is a viral antigen, bacterial antigenor parasitic antigen.

In some embodiments of any of the embodiments herein, the recombinantreceptor is a transgenic T cell receptor (TCR) or a functional non-Tcell receptor.

In some embodiments of any of the embodiments herein, the recombinantreceptor is a chimeric receptor, which optionally is a chimeric antigenreceptor (CAR).

In some embodiments of any of the embodiments herein, the inhibitor is aselective inhibitor of the target protein tyrosine kinase; and/or theinhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) that is at least 10 or atleast 100 times lower than that of the IC50 of the inhibitor for anyprotein tyrosine kinase or TEC family kinase distinct from the targetprotein tyrosine kinase, and/or inhibits the target protein tyrosinekinase with an IC50 at least 2, at least 10 or at least 100 times lowerthan that the IC50 value of the inhibitor for both ITK and BTK; and/orthe inhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) of less than or less thanabout 1000 nM, 900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM,100 nM or less.

In some embodiments of any of the kits herein, the inhibitor is a smallmolecule, peptide, protein, antibody or antigen-binding fragmentthereof, an antibody mimetic, an aptamer, or a nucleic acid molecule. Insome embodiments of any of the embodiments herein, the inhibitor isselected from Formula (II), ONO/GS-4059, Compound 30 or Compound 38,GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;CNX-774; and LFM-A13.

In some embodiments of any of the kits herein, the inhibitor containsthe compound of Formula (II), or an enantiomer,pharmaceutically-acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof or a pharmaceutical composition comprisingany of the foregoing.

In some embodiments of any of the kits herein, the instructions are foradministering the inhibitor concurrently with or subsequently toinitiation of administration of the composition comprising the T cells.In some embodiments of any of the kits herein, the instructions are foradministering the inhibitor subsequently to initiation of administrationof the T cells.

In some embodiments of any of the kits herein, the instructions are foradministering the inhibitor within, or within about, 1 hour, 2 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours or 1 week of theinitiation of the administration of the T cells.

In some embodiments of any of the kits herein, the instructions are foradministering the inhibitor at a time in which: the number of cells ofthe T cell therapy detectable in the blood from the subject is decreasedcompared to in the subject at a preceding time point after initiation ofthe administration of the T cells; the number of cells of the T celltherapy detectable in the blood is less than or less than about1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold or 100-foldor less the peak or maximum number of the cells of the T cell therapydetectable in the blood of the subject after initiation ofadministration of the administration of the T cells; and/or at a timeafter a peak or maximum level of the cells of the T cell therapy aredetectable in the blood of the subject, the number of cells of orderived from the T cells detectable in the blood from the subject isless than less than 10%, less than 5%, less than 1% or less than 0.1% oftotal peripheral blood mononuclear cells (PBMCs) in the blood of thesubject.

In some embodiments of any of the embodiments herein, the increase ordecrease is by greater than or greater than about 1.2-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more.

In some embodiments of any of the embodiments herein, the instructionsare for administering the inhibitor for a time period up to 2 days, upto 7 days, up to 14 days, up to 21 days, up to one month, up to twomonths, up to three months, up to 6 months or up to 1 year afterinitiation of the administration of the administration of the T cells.

In some embodiments of any of the embodiments herein, the instructionsare for further administering the inhibitor from at least afterinitiation of administration of the T cells, until:

-   -   the number of cells of or derived from the T cells administered        detectable in the blood from the subject is increased compared        to in the subject at a preceding time point just prior to        administration of the inhibitor or compared to a preceding time        point after administration of the T-cell therapy; the number of        cells of or derived from the T cells detectable in the blood is        within 2.0-fold (greater or less) the peak or maximum number        observed in the blood of the subject after initiation of        administration of the T cells; the number of cells of the T        cells detectable in the blood from the subject is greater than        or greater than about 10%, 15%, 20%, 30%, 40%, 50%, or 60% total        peripheral blood mononuclear cells (PBMCs) in the blood of the        subject; and/or the subject exhibits a reduction in tumor burden        as compared to tumor burden at a time immediately prior to the        administration of the T cells or at a time immediately prior to        the administration of the inhibitor; and/or the subject exhibits        complete or clinical remission.

In some embodiments of any of the embodiments herein, the geneticallyengineered T cells contain cells that are autologous to the subject. Insome embodiments of any of the embodiments herein, the geneticallyengineered T cells contain T cells that are allogeneic to the subject.

Provided in some aspects are methods of engineering immune cellsexpressing a recombinant receptor, comprising: contacting a populationof cells comprising T cells with an inhibitor of a target proteintyrosine kinase, wherein the inhibitor does not inhibit ITK and/orinhibits ITK with a half-maximal inhibitory concentration (IC50) ofgreater than or greater than about 1000 nM and/or the target proteintyrosine kinase is a tyrosine kinase expressed in hepatocellularcarcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a BMX/ETK, or anERBB4; and introducing a nucleic acid encoding a recombinant receptorinto the population of T cells under conditions such that therecombinant receptor is expressed.

In some embodiments of any of the methods herein, the population ofcells is or contains T cells, optionally CD4+ or CD8+.

In some embodiments of any of the methods herein, the population ofcells are isolated from a subject, optionally a human subject.

In some embodiments of any of the methods herein, the contacting occursprior to and/or during the introducing.

Provided in some aspects are methods of producing genetically engineeredT cells, comprising introducing a nucleic acid molecule encoding arecombinant receptor into a primary T cell, wherein the T cells is froma subject having been administered an inhibitor of a target proteintyrosine kinase, wherein the inhibitor does not inhibit ITK and/orinhibits ITK with a half-maximal inhibitory concentration (IC50) ofgreater than or greater than about 1000 nM and/or the target proteintyrosine kinase is a tyrosine kinase expressed in hepatocellularcarcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a BMX/ETK, or anERBB4.

In some embodiments of any of the methods herein, the subject has beenadministered the inhibitor no more than 30 days, 20 days, 10 days, 9days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 dayprior to introducing the nucleic acid molecule.

In some embodiments of any of the methods herein: the inhibitor is aselective inhibitor of the target protein tyrosine kinase; and/or theinhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) that is at least 10 or atleast 100 times lower than that of the IC50 of the inhibitor for anyprotein tyrosine kinase or TEC family kinase distinct from the targetprotein tyrosine kinase, and/or inhibits the target protein tyrosinekinase with an IC50 at least 2, at least 10 or at least 100 times lowerthan that the IC50 value of the inhibitor for both ITK and BTK; and/orthe inhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC50) of less than or less thanabout 1000 nM, 900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM,100 nM or less.

In some embodiments of any of the methods herein, the inhibitor is asmall molecule, peptide, protein, antibody or antigen-binding fragmentthereof, an antibody mimetic, an aptamer, or a nucleic acid molecule.

In some embodiments of any of the methods herein, the inhibitor isselected from the compound of Formula (II), ONO/GS-4059, Compound 30 orCompound 38, GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292;ONO-4059; CNX-774; and LFM-A13.

In some embodiments of any of the methods herein, the inhibitor containsthe compound of Formula (II), or an enantiomer,pharmaceutically-acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof or a pharmaceutical composition comprisingany of the foregoing.

In some embodiments of any of the methods herein, the T cells containCD4+ or CD8+ cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows graphs of normalized target cell numbers assessingtarget-specific cytolytic activity in triplicate wells co-cultured withCAR T cells with ibrutinib or the compound of Formula (II) (mean±SEM).

FIG. 1B shows a representative image of target cells (NucLight RedK562.CD19 cells) co-cultured with CAR T cells at an effector to targetratio (E:T) of 2.5:1 at the start and end of the cytotoxic assay.

FIG. 1C and FIG. 1D shows the results of a study assessingtarget-specific cytolytic activity of anti-CD19 CAR T cells in thepresence or absence of ibrutinib or the compound of Formula (II),respectively. The graphs show data from three independent donors and arenormalized to untreated control (100%). The mean±SEM are depicted andstatistically significant differences are indicated P<0.00001 (****).

FIG. 2A and FIG. 2B show CART cell expression of CD25, CD28, CD39 andCD95 following culture of CD4+ and CD8+ cells in the presence or absenceof indicated concentrations of ibrutinib or the compound of Formula(II), respectively.

FIG. 2C and FIG. 2D show representative results of CAR T cell from onedonor-derived cells for the percentage of TCM (CCR7+CD45RA−) and TEM(CCR7-CD45RA−) over four days after initial stimulation in the presenceof ibrutinib or the compound of Formula (II), respectively.

FIG. 2E and FIG. 2F show CAR-T cell expression of CD69, CD107a and PD-1following culture of CD4+ and CD8+ cells, respectively, in the presenceor absence of indicated concentrations of ibrutinib.

FIG. 2G and FIG. 2H show CAR-T cell expression of CD69, CD107a and PD-1following culture of CD4+ and CD8+ cells, respectively, in the presenceor absence of indicated concentrations of the compound of Formula (II).

FIG. 3A and FIG. 3B shown measurement of IFN-gamma, IL-2 and TNF-alphafrom supernatant of CAR T cells from one donor-derived cells stimulatedwith target cells at an E:T of 2.5:1 and treated with ibrutinib or thecompound of Formula (II), respectively.

FIG. 3C depicts percentage change in a readout of secreted cytokine,IFN-gamma, IL-2 and TNF-alpha, after stimulation of CAR-T cells for 2days in the presence of ibrutinib compared to untreated controls, incells derived from three donors, in two 2 independent assays.

FIG. 3D depicts percentage change in a readout of secreted cytokine,IFN-gamma, IL-2 and TNF-alpha, after stimulation of CAR-T cells for 2days in the presence of the compound of Formula (II) compared tountreated controls, for three donors in 2 independent assays.

FIG. 4A shows the number of population doublings of CAR-T cells afterindividual round of restimulation in a serial stimulation assay, in theabsence (control) or presence of 50 nM or 500 nM ibrutinib. Arrowsindicate the time point of each re-stimulation where CAR T cells werecounted and new target cells along with ibrutinib were added.

FIG. 4B shows the number of population doublings of CAR-T cells afterindividual round of restimulation in a serial stimulation assay, in theabsence (control) or presence of 158 nM or 1581 nM of the compound ofFormula (II). Arrows indicate the time point of each re-stimulationwhere CAR T cells were counted and new target cells along with thecompound of Formula (II) were added.

FIG. 4C shows the number of cells at day 18 after 5 rounds ofrestimulation in the presence or absence of the indicated concentrationsof ibrutinib in a serial stimulation assay, P<0.05 (*)

FIG. 4D shows the number of cells at day 18 after 5 rounds ofrestimulation in the presence or absence of the indicated concentrationsof the compound of Formula (II) in a serial stimulation assay, P<0.05(*).

FIG. 5A shows the results of a flow cytometry study assessing surfaceexpression levels of cell surface markers used to differentiate TH2versus TH1 phenotype T cells, following stimulation of T cells in thepresence of the compound of Formula (II) or ibrutinib.

FIG. 5B shows the percentage of TH1 cells observed over time, asmeasured by the flow cytometry assay, for T cells cultured in thepresence or absence of the compound of Formula (II) or ibrutinib.

FIG. 5C shows the percentage of TH1 cells, as measured by the flowcytometry assay, at day 18 following serial restimulation of T cells inthe absence or presence of the indicated concentrations of the compoundof Formula (II) or ibrutinib.

FIG. 5D shows expression of CD25, CD38, CD39 and CD45RO (FIG. 5D) atdays 0, 11, 18 and 21 of serial stimulation in the presence of thecompound of Formula (II). Representative results from CAR T cells fromone donor-derived cells are shown.

FIG. 5E shows expression of CD62L, CD69, CD107a and PD-1 (FIG. 5E) atdays 0, 11, 18 and 21 of serial stimulation in the presence of thecompound of Formula (II). Representative results from CAR T cells fromone donor-derived cells are shown.

FIG. 5F and FIG. 5G expression of CD25, CD38, CD39 and CD45RO (FIG. 5D)and CD62L, CD69, CD107a and PD-1 (FIG. 5E) at days 0, 11, 18 and 21 ofserial stimulation in the presence of ibrutinib. Representative resultsfrom CAR T cells from one donor-derived cells are shown.

FIG. 6A shows results of a study assessing tumor burden over time in adisseminated tumor xenograft mouse model (the model identified as beingresistant to BTK inhibition), following administration of a sub-optimaldose of CAR+ T cells (Anti-CD19 CAR), in combination with oraladministration of (a) vehicle alone (P.O. vehicle) or (b) ibrutinib(P.O. Ibrutinib), (iii) oral administration of vehicle alone (vehicle)or (iv) oral administration of ibrutinib alone (ibrutinib). FIG. 6Bshows results of the same study at greater time points after post-tumorrejection in mice that were treated with CAR+ T cells from two differentdonor-derived cells in the presence or absence of ibrutinib or vehiclecontrol. The results in FIG. 6A and FIG. 6B depict tumor growth overtime as indicated by measuring average radiance by bioluminescence.

FIG. 6C shows a Kaplan Meier curve for survival of the animals in thesetreatment groups described with reference to FIG. 6A. FIG. 6D showsresults of survival in the same study at greater time points afterpost-tumor rejection in mice that were treated with CAR+ T cells fromtwo different donor-derived cells in the presence or absence ofibrutinib or vehicle control.

FIG. 7A shows a Kaplan meier curve depicting observed survival oftumor-bearing mice administered CAR-T cells generated from two differentdonors, alone or in combination with administration of daily ibrutinibor the compound of Formula (II), each administered via drinking water.Statistically significant differences are shown, P<0.001 (***).

FIG. 7B shows tumor growth over time as indicated by measuring averageradiance by bioluminescence from mice administered CAR-T cells generatedfrom two different donors and treated with ibrutinib or the compound ofFormula (II), each administered via drinking water. Statisticallysignificant differences are shown, two-way ANOVA P<0.05 (*), P<0.01(**).

FIG. 7C shows results of a study assessing numbers of CAR-T cells in theblood and bone marrow of mice treated with or without ibrutinib.Statistically significant differences are indicated as P<0.05 (*),P<0.001 (***).

FIG. 7D shows the number of cells in the blood at day 19 post CAR-T celltransfer after treatment or with or without ibrutinib. Statisticallysignificant differences are indicated as P<0.05 (*).

FIG. 7E shows results of a study assessing numbers of CAR-T cells in theblood and bone marrow of mice treated with or without the compound ofFormula (II). Statistically significant differences are indicated asP<0.001 (***) and P<0.0001 (****).

FIG. 7F shows the number of cells in the blood at day 19 post CAR-T celltransfer after treatment or with or without the compound of Formula(II). Statistically significant differences are indicated as P<0.001(***).

FIG. 7G shows the tumor cell count at days 7, 12, 19 and 26 post CAR Ttransfer in the blood and bone marrow of mice treated with CAR-T cellsalone or with ibrutinib. Statistically significant differences areindicated as P<0.05 (*), P<0.01 (**), P<0.001 (***) and P<0.0001 (****).

FIG. 7H shows the tumor cell count at days 7, 12, 19 and 26 post CAR Ttransfer in the blood, bone marrow of mice treated with CAR-T cellsalone or with the compound of Formula (II). Statistically significantdifferences are indicated as P<0.05 (*), P<0.01 (**), P<0.001 (***) andP<0.0001 (****).

FIG. 8A depicts T-distributed stochastic neighbor embedding (t-SNE) highdimensional analysis of surface markers on CAR-engineered T cellsharvested from the bone marrow of animals at day 12 after treatment withthe CAR-T cells either in the absence of inhibitor (control) or in thepresence of ibrutinib or the compound of Formula (II).

FIG. 8B depicts four populations derived from T-distributed stochasticneighbor embedding (t-SNE) high dimensional analysis of surface markerson CAR-engineered T cells harvested from the bone marrow of animals atday 12 after treatment with the CAR-T cells either in the absence ofinhibitor (control) or in the presence of ibrutinib or the compound ofFormula (II). The results represent pooled analysis from three mice pergroup.

FIG. 8C depicts histograms showing the individual expression profiles ofCD4, CD8, CD62L, CD45RA, CD44 and CXCR3 from the 4 gated t-SNE overlaidon the expression of the total population (shaded histogram).

FIG. 8D depicts the percentage and fold change of each t-SNE populationfrom control mice or mice treated with ibrutinib or the compound ofFormula (II).

FIG. 9A shows the number of population doublings in a serial stimulationassay over a 21 day culture period of CAR-engineered cells, generatedfrom cells obtained from subjects with diffuse large B-cell lymphoma(DLBCL), with cells cultured in the absence of ibrutinib (control) or inthe presence of 50 nM or 500 nM ibrutinib. Arrows indicate the timepoint of each re-stimulation where CAR T cells were counted and newtarget cells along with ibrutinib was added.

FIG. 9B shows results of an assay for cytolytic activity of the CAR-Tcells for CD19-expressing target cells after 16 days of serialrestimulation in the presence or absence of ibrutinib. Percent killingwas normalized to untreated control (100%). Data shown as mean±SEM fromreplicate wells. Statistically significant differences are indicated asP<0.001 (***), P<0.0001 (****).

FIG. 10A is a Volcano plot depicting differentially expressed genes fromday 18 serially stimulated CAR T cells treated with 500 nM ibrutinibcompared with control. Significantly differentially upregulated genesare on the right side of right dashed line and significantlydifferentially downregulated genes are on left side of left dashed line(FDR<0.05, abslog2FC>0.5).

FIG. 10B depicts a Volcano plot of expressed genes from day 18 seriallystimulated CAR T cells treated with 1581 nM of the compound of Formula(II) compared with control. Significantly differentially upregulatedgenes are on the right side of right dashed line and significantlydifferentially downregulated genes are on left side of left dashed line(FDR<0.05, abslog2FC>0.5).

FIG. 10C is a heat map depicting normalized expression across 3 donors(mean Transcripts per Million per donor+condition, z-score normalizedper gene) of the differentially expressed genes from FIG. 10A in thecontrol and 500 nM ibrutinib groups.

FIG. 11A-11B depict the expression (TPM, transcrips per million) boxplot profiles of GZMA and SELL (CD62L) summarized across donors andexperiments per condition from serially stimulated CAR T cells treatedwith 50 nM or 500 nM ibrutinib compared with control.

FIG. 12A is a representative histogram of CD62L expression in CAR Tcells from one donor-derived cells after 18 days of serial stimulation,as measured by flow cytometry.

FIG. 12B depicts the fold change in the percentage of CD62L+ CART cellsfrom one donor-derived cells after 18 days of serial stimulationnormalized to control, as measured by flow cytometry. The data are fromtwo independent experiments (mean±SEM).

DETAILED DESCRIPTION

Provided herein are methods of enhancing or modulating proliferationand/or activity of T cell activity associated with administration of animmunotherapy or immunotherapeutic agent, such as a compositionincluding cells for adoptive cell therapy, e.g., such as a T celltherapy (e.g. CAR-expressing T cells) or a T cell-engaging therapeuticagent, such as a bispecific or multispecific agent or antibody, capableof recruiting one or more T cells or other immune cells. In someembodiments, the combination therapy involves administration of aninhibitor of a target protein tyrosine kinase that is not IL-2-inducibleT cell kinase (ITK) and/or in which the target protein tyrosine kinaseis one or more protein-tyrosine kinase selected from Bruton's tyrosinekinase (Btk), tec protein tyrosine kinase (TEC), BMX non-receptortyrosine kinase (Etk), TXK tyrosine kinase (TXK) and/or receptortyrosine-protein kinase ErbB4 (ErbB4), e.g. the compound of Formula (II)the compound of Formula (II), and administration of the immunotherapy orimmunotherapeutic agent, such as a composition including cells foradoptive cell therapy, e.g., such as a T cell therapy (e.g.CAR-expressing T cells) or a T cell-engaging therapeutic agent.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section heading used herein are for organizational purposes only andare not to be construed as limiting the subject matter described.

I. Overview

Provided herein are combination therapies involving administration of acell therapy or other immunotherapeutic agent that contains or modulatesthe engagement or activity of T cells, in combination with an inhibitor.Among such agents are agents for T cell therapy, such engineered T cells(e.g., CAR-expressing T cells) and other agents for use in adoptive Tcell therapy. The inhibitors generally are inhibitors of target kinases,such as target protein tyrosine kinases, including non-receptor tyrosinekinases such as those of the TEC family of kinases. In some aspects, theinhibitor is an inhibitor of a protein tyrosine kinase that is notIL-2-inducible T cell kinase (ITK) and/or does not inhibit ITK. In someembodiments, the target protein tyrosine kinase is one or more ofBruton's tyrosine kinase (Btk), tec protein tyrosine kinase (TEC), BMXnon-receptor tyrosine kinase (Etk), TXK tyrosine kinase (TXK) and/orreceptor tyrosine-protein kinase ErbB4 (ErbB4), Among the inhibitors arethe compound of Formula (II).

T cell-based therapies, such as adoptive T cell therapies (includingthose involving the administration of cells expressing chimericreceptors specific for a disease or disorder of interest, such aschimeric antigen receptors (CARs) and/or other recombinant antigenreceptors, as well as other adoptive immune cell and adoptive T celltherapies) can be effective in the treatment of cancer and otherdiseases and disorders. The engineered expression of recombinantreceptors, such as chimeric antigen receptors (CARs), on the surface ofT cells enables the redirection of T-cell specificity. In clinicalstudies, CAR-T cells, for example anti-CD19 CAR-T cells, have produceddurable, complete responses in both leukemia and lymphoma patients(Porter et al. (2015) Sci Transl Med., 7:303ra139; Kochenderfer (2015)J. Clin. Oncol., 33: 540-9; Lee et al. (2015) Lancet, 385:517-28; Maudeet al. (2014) N Engl J Med, 371:1507-17).

In certain contexts, available approaches to adoptive cell therapy maynot always be entirely satisfactory. In some contexts, optimal efficacycan depend on the ability of the administered cells to recognize andbind to a target, e.g., target antigen, to traffic, localize to andsuccessfully enter appropriate sites within the subject, tumors, andenvironments thereof. In some contexts, optimal efficacy can depend onthe ability of the administered cells to become activated, expand, toexert various effector functions, including cytotoxic killing andsecretion of various factors such as cytokines, to persist, includinglong-term, to differentiate, transition or engage in reprogramming intocertain phenotypic states (such as long-lived memory,less-differentiated, and effector states), to avoid or reduceimmunosuppressive conditions in the local microenvironment of a disease,to provide effective and robust recall responses following clearance andre-exposure to target ligand or antigen, and avoid or reduce exhaustion,anergy, peripheral tolerance, terminal differentiation, and/ordifferentiation into a suppressive state.

In some cases, responses can be improved by administration orpreconditioning with a lymphodepleting therapy, which in some aspectsincreases the persistence and/or efficacy of the cells followingadministration, as compared to methods in which the preconditioning isnot carried out or is carried out using a different lymphodepletingtherapy. The lymphodepleting therapy generally includes theadministration of fludarabine, typically in combination with anotherchemotherapy or other agent, such as cyclophosphamide, which may beadministered sequentially or simultaneously in either order. In a recentphase I/II clinical study, complete response (CR) in acute lymphoblasticleukemia (ALL), non-Hodgkin's lymphoma (NHL) and chronic lymphocyticleukemia (CLL) patients was 94%, 47% and 50% respectively, and diseasefree survival rates were greater in patients that receivedcyclophosphamide and fludarabine lymphodepletion compared to those whoreceived cyclophosphamide but not fludarabine (Cameron et al. (2016) JClin Oncol, 34 (suppl; abstr 102). In some aspects, however, even withlymphodepleting therapies, CAR-T cell therapies are not alwaysconsistently effective in all subjects.

In some aspects, the provided methods and uses provide for or achieveimproved or more durable responses or efficacy as compared to certainalternative methods, such as in particular groups of subjects treated.In some embodiments, the methods are advantageous by virtue ofadministering an immunotherapy or immunotherapeutic agent, such as acomposition including cells for adoptive cell therapy, e.g., such as a Tcell therapy (e.g. CAR-expressing T cells) or a T cell-engagingtherapeutic agent, such as a bispecific or multispecific agent orantibody, and an inhibitor of a target protein-tyrosine kinase thatinhibits, targets or reduces the activity of a target protein tyrosinekinase other than IL-2-inducible T cell kinase (ITK), such as one ormore of Bruton's tyrosine kinase (Btk), tec protein tyrosine kinase(TEC), BMX non-receptor tyrosine kinase (Etk), TXK tyrosine kinase (TXK)and/or receptor tyrosine-protein kinase ErbB4 (ErbB4) e.g. the compoundof Formula (II).

The provided methods are based on observations that BTK inhibitors, e.g.ibrutinib and Formula (II), improve T cell function, including functionsrelated to the expansion, proliferation and persistence of T cells. Itis found herein that this effect, and extent and degree of this effect,is substantially the same whether using ibrutinib or the compound ofFormula (II). Yet, ibrutinib and the compound of Formula (II) do notcompletely exhibit the same specificity for protein tyrosine kinases.Ibrutinib is an irreversible small molecule inhibitor (SMI) that blocksthe activity of Bruton's tyrosine kinase (Btk) and also exhibitsactivity on ITK and numerous other TEC family kinases and other kinases.The compound of Formula (II) was developed as the next generation of Btkinhibitor with greater specificity and potency compared to ibrutinib (Wuet al. (2016) J Hematol Oncol, 9:21; Byrd et al. (2016) N Engl J Med.,374:323-332). Of interest, the compound of Formula (II) does not exhibitactivity towards interleukin-2-inducible kinase (ITK), which is a kinasethat is highly expressed in both CD4 and CD8 T cells and believed to beinvolved in effects on downstream T cell receptor signaling (Berg et al.(2005) Annu Rev. Immunol., 23:549-600). Thus, this finding indicatesthat the effect of the inhibitors on T cell function is not mediated byITK but is mediated by inhibition of one or more of the other kinasestargeted by these inhibitors, such as one or more of BTK, TEC, BMX/ETK,RLK/TXK and/or ERBB4.

BTK inhibitors are generally used for the treatment of B cellmalignancies. For example, ibrutinib, is approved for use in mantle celllymphoma (MCL) and Waldenström's Macroglobulinemia in the relapsedrefractory setting (Davids et al. (2014) Future Oncol., 10:957-67). Insome cases, aberrant activation of the B-cell receptor (BCR) signalingpathway is the main mechanism underlying B cell malignancies such as MCLand CLL, whereby chronic Btk signaling can initiate a phosphorylationcascade through NF-kB and MAP kinases promoting B cell survival andaberrant activation. Thus, existing methods of employing BTK inhibitors,e.g. ibrutinib, are used for treating B cell malignancies.

The provided findings indicate that combination therapy of the inhibitorin methods involving T cells, such as involving administration ofadoptive T cell therapy, achieves improved function of the T celltherapy. In some embodiments, combination of the cell therapy (e.g.,administration of engineered T cells) with the inhibitor of one or moretarget protein tyrosine kinase from among BTK, TEC, BMX/ETK, RLK/TXKand/or ERBB4 (such as a selective and/or irreversible inhibitor of suchkinase), improves or enhances one or more functions and/or effects ofthe T cell therapy, such as persistence, expansion, cytotoxicity, and/ortherapeutic outcomes, e.g., ability to kill or reduce the burden oftumor or other disease or target cell. In some embodiments, an inhibitorof one or more target protein tyrosine kinase from among BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 (such as a selective and/or irreversibleinhibitor of such kinase) may dampen CAR T activation at higherconcentrations while increasing activation at lower concentrations.

In some aspects, such effects are observed despite that the tumor ordisease or target cell itself is insensitive to the inhibitor, toinhibitors targeting the kinase to which the inhibitor is selective,and/or is resistant to the inhibition of BTK, TEC, BMX/ETK, RLK/TXKand/or ERBB4 by the inhibitor. For example, in some embodiments, thecancer is insensitive to or has become resistant to the inhibitor, or toinhibition of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4 by the inhibitor,e.g., by the compound of Formula (II). In some embodiments, thecombination with the inhibitor, while improving one or more outcomes orfunctional attributes, does not affect one or more side effects orunwanted changes in the T cells, such as does not reduce the ability ofthe cells to become activated, secrete one or more desired cytokines,expand and/or persist, e.g., as measured in an in vitro assay ascompared to such cells cultured under conditions otherwise the same butin the absence of the inhibitor. Thus in some embodiments, provided aremethods and combinations that result in improvements in T cell functionor phenotype, e.g., in intrinsic T cell functionality and/or intrinsic Tcell phenotype, generally without compromising one or more other desiredproperties of functionality, e.g., of CAR-T cell functionality.

Hence, in some embodiments, the provided methods can potentiate CAR-Tcell therapy, which, in some aspects, can improve outcomes for treatmentof subjects that have a cancer that is resistant or refractory to othertherapies, is an aggressive or high-risk cancer, and/or that is or islikely to exhibit a relatively lower response rate to a CAR-T celltherapy administered without the inhibitor compared to another type ofcancer.

In some embodiments, the methods can be used for treating B cellmalignancies or hematological malignancies, and in particular suchmalignancies in which responses, e.g. complete response, to treatmentwith the immunotherapy or immunotherapeutic agent, such as a compositionincluding cells for adoptive cell therapy, e.g., such as a T celltherapy (e.g. CAR-expressing T cells) or a T cell-engaging therapeuticagent, alone is relatively low compared to other B cell malignancies(e.g. a CR in a less than or less than about 60%, less than about 50% orless than about 45% of the subjects so treated) and/or in which thesubject is not responsive to treatment with the inhibitor alone. In someembodiments, the combination therapy provided herein is for use in asubject having a cancer in which the subject and/or the cancer isresistant to inhibition of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4 orcomprises a population of cells that are resistant to inhibition by theinhibitor. In some embodiments, the combination therapy provided hereinis for use in a subject having a cancer in which the subject and/or thecancer comprises a mutation or disruption in a nucleic acid encodingBTK, in which such mutation is capable of reducing or preventinginhibition of the BTK by the inhibitor, e.g. the compound of Formula(II).

In some embodiments, the combination therapy provided herein is for usein a subject having a cancer in which at the time of administration ofthe immunotherapy or immunotherapeutic agent, such as a compositionincluding cells for adoptive cell therapy, e.g., such as a T celltherapy (e.g. CAR-expressing T cells) or a T cell-engaging therapeuticagent, and at the time of administering the inhibitor, the subject hasrelapsed following remission after treatment with, or been deemedrefractory to treatment with the inhibitor and/or with a BTK inhibitortherapy.

In some embodiments, certain cancers, such as NHL, e.g. high-risk oraggressive NHL, such as DLBCL, and/or chronic lymphocytic leukemia (CLL)can be associated with defects in or reduction in intrinsic T cellfunctionality, which, in some cases, is influenced by the diseaseitself. For example, the pathogenesis of many cancers, such as CLL andNHL, e.g. DLBCL, can be associated with immunodeficiency, leading topromotion of tumor growth and immune evasion, such as due toimmunosuppression of T cells, e.g. driven by one or more factors in thetumor microenvironment. In some cases, alleviating intrinsic T celldefects obtained from cancers of such patients for use in connectionwith adoptive cell therapy could provide for more potent responses toadoptive T cell therapy, e.g. CAR-T cell therapy.

In some embodiments, the provided methods are for treating a cancer in asubject in which such subject's T cells display or have been observed todisplay a decreased level of a factor indicative of T cell function,health, or activity, as compared to a reference population of T cells ora reference or threshold level, e.g. T cells from a subject not havingor suspected of having a cancer, such as from a healthy or normalsubject. In some embodiments, the provided methods are for treatingsubjects identified as having high-risk NHL and/or aggressive NHL,diffuse large B cell lymphoma (DLBCL), primary mediastinal large B celllymphoma (PMBCL), T cell/histocyte-rich large B cell lymphoma (TCHRBCL),Burkitt's lymphoma, mantle cell lymphoma (MCL), and/or follicularlymphoma (FL). For example, as shown herein, in the presence of theexemplary BTK inhibitor ibrutinib, T cells engineered from subjectshaving DLBCL exhibit a greater T cell functional activity, indicatingthat the function of the T cells is potentiated in the presence of theinhibitor. In some embodiments, the subject has DLBCL. In someembodiments, the provided methods are for treating a subject havingchronic lymphocytic leukemia (CLL). In some embodiments of such methods,the administered engineered T cells are autologous to the subject.

In some embodiments, the provided methods also include methods in whichthe cancer is not a B cell malignancy, is not a B cell leukemia orlymphoma, is a non-hematologic cancer or is a solid tumor; and/or theantigen is not a B cell antigen, such as is not CD19, CD20, CD22, andROR1. In some embodiments, the combination therapy includesadministration to a subject with a solid tumor, such as a sarcoma orcarcinoma, 1) T cells that specifically recognize and/or target anantigen associated with the cancer and/or present on a universal tag and2) an inhibitor of a target protein tyrosine kinase that is other thanITK, such as is one or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4,e.g. the compound of Formula (II). In some embodiments, the antigenrecognized or targeted by the T cells is Her2, L1-CAM, mesothelin, CEA,hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD38,CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR vIII,FBP, FCRLS, FCRHS, fetal acethycholine e receptor, GD2, GD3, HMW-MAA,IL-22R-alpha, IL-13R-alpha2, kdr, Lewis Y, L1-cell adhesion molecule(L1-CAM), Melanoma-associated antigen (MAGEMAGE-A1, MAGE-A3, MAGE-A6,Preferentially expressed antigen of melanoma (PRAME), survivin, EGP2,EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, GD3, HMW-MAA,CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folatereceptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors,5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, and an antigenassociated with a universal tag, a cancer-testes antigen, mesothelin,MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetalantigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostatespecific antigen, PSMA, estrogen receptor, progesterone receptor,ephrinB2, CD123, c-Met, GD-2 O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1(WT-1), a cyclin, cyclin A2, CCL-1, CD138, or a pathogen-specificantigen.

In some embodiments of the provided methods, one or more properties ofadministered genetically engineered cells can be improved or increasedor greater compared to administered cells of a reference composition,such as increased or longer expansion and/or persistence of suchadministered cells in the subject or an increased or greater recallresponse upon restimulation with antigen. In some embodiments, theincrease can be at least a 1.2-fold, at least 1.5-fold, at least 2-fold,at last 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, or at least 10-foldincrease in such property or feature compared to the same property orfeature upon administration of a reference cell composition. In someembodiments, the increase in one or more of such properties or featurescan be observed or is present within one months, two months, threemonths, four months, five months, six months, or 12 months afteradministration of the genetically engineered cells.

In some embodiments, a reference cell composition can be a compositionof T cells from the blood of a subject not having or not suspected ofhaving the cancer or is a population of T cells obtained, isolated,generated, produced, incubated and/or administered under the same orsubstantially the conditions, except not having been incubated oradministered in the presence of an inhibitor of a target proteintyrosine kinase that is other than ITK, such as is one or more of BTK,TEC, BMX/ETK, RLK/TXK and/or ERBB4. In some embodiments, the referencecell composition contains genetically engineered cells that aresubstantially the same, including expression of the same recombinantreceptor, e.g. CAR. In some aspects, such T cells are treatedidentically or substantially identically, such as manufacturedsimilarly, formulated similarly, administered in the same or about thesame dosage amount and other similar factors.

In some embodiments, a genetically engineered cell with increasedpersistence exhibit better potency in a subject to which it isadministered. In some embodiments, the persistence of geneticallyengineered cells, such as CAR-expressing T cells, in the subject uponadministration is greater as compared to that which would be achieved byalternative methods, such as those involving administration of areference cell composition. In some embodiments, the persistence isincreased at least or about at least 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more.

In some embodiments, the degree or extent of persistence of administeredcells can be detected or quantified after administration to a subject.For example, in some aspects, quantitative PCR (qPCR) is used to assessthe quantity of cells expressing the recombinant receptor (e.g.,CAR-expressing cells) in the blood or serum or organ or tissue (e.g.,disease site) of the subject. In some aspects, persistence is quantifiedas copies of DNA or plasmid encoding the receptor, e.g., CAR, permicrogram of DNA, or as the number of receptor-expressing, e.g.,CAR-expressing, cells per microliter of the sample, e.g., of blood orserum, or per total number of peripheral blood mononuclear cells (PBMCs)or white blood cells or T cells per microliter of the sample. In someembodiments, flow cytometric assays detecting cells expressing thereceptor generally using antibodies specific for the receptors also canbe performed. Cell-based assays may also be used to detect the number orpercentage of functional cells, such as cells capable of binding toand/or neutralizing and/or inducing responses, e.g., cytotoxicresponses, against cells of the disease or condition or expressing theantigen recognized by the receptor. In any of such embodiments, theextent or level of expression of another marker associated with therecombinant receptor (e.g. CAR-expressing cells) can be used todistinguish the administered cells from endogenous cells in a subject.

Also provided are methods for engineering, preparing, and producing thecells, compositions containing the cells and/or inhibitor, and kits anddevices containing and for using, producing and administering the cellsand/or inhibitor, such as in accord with the provided combinationtherapy methods.

II. Combination Therapy

Provided herein are methods for combination therapy for treating adisease or disorder, e.g. a cancer or proliferative disease, thatincludes administering to a subject a combination therapy of 1) aninhibitor of a target protein tyrosine kinase that does not inhibit ITKand/or that inhibits one or more of BTK, TEC, BMX/ETK, RLK/TXK and/orERBB4 and 2) an immunotherapy or immunotherapeutic agent, such as anadoptive immune cell therapy, e.g. T cell therapy (e.g. CAR-expressingcell, e.g. T cells) or a T-cell engaging or immune modulatory therapy,e.g. a multispecific T cell recruiting antibody and/or checkpointinhibitor. In some embodiments, the immunotherapy is an adoptive immunecell therapy comprising T cells that specifically recognize and/ortarget an antigen associated with a disease or disorder, e.g. a canceror proliferative disease. Also provided are combinations and articles ofmanufacture, such as kits, that contain a composition comprising the Tcell therapy and/or a composition comprising the inhibitor, such as aninhibitor of one or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4, anduses of such compositions and combinations to treat or prevent diseases,conditions, and disorders, including cancers. Such methods can includeadministration of the inhibitor prior to, simultaneously with, during,during the course of (including once and/or periodically during thecourse of), and/or subsequently to, the administration (e.g., initiationof administration) of the T cell therapy (e.g. CAR-expressing T cells)or other therapy such as the T cell-engaging therapy. In someembodiments, the administrations can involve sequential or intermittentadministrations of the inhibitor and/or the immunotherapy orimmunotherapeutic agent, e.g. T cell therapy.

In some embodiments, the cell therapy is adoptive cell therapy. In someembodiments, the cell therapy is or comprises a tumor infiltratinglymphocytic (TIL) therapy, a transgenic TCR therapy or arecombinant-receptor expressing cell therapy (optionally T celltherapy), which optionally is a chimeric antigen receptor(CAR)-expressing cell therapy. In some embodiments, the therapy targetsCD19 or is a B cell targeted therapy. In some embodiments, the cells anddosage regimens for administering the cells can include any as describedin the following subsection A under “Administration of Cells.”

In some embodiments, the inhibitor selectively inhibits one or moretarget protein tyrosine kinase from among BTK, TEC, BMX/ETK, RLK/TXKand/or ERBB4 compared to other protein tyrosine kinases or compared toother TEC family kinases. In some embodiments, the inhibitor does notinhibit ITK or has an IC50 or Kd for ITK of greater than 1000 nM and/orselectively inhibits one or more of BTK, TEC, BMX/ETK, RLK/TXK and/orERBB4 compared to ITK. In some embodiments, the inhibitor exhibits atleast 1.5-fold, 2.5-fold, 5.0-fold, 10.0-fold, 25-fold, 50-fold,100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or moreactivity for one or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4compared to ITK. In some embodiments, the inhibitor selectively inhibitsone of TEC, TEC, BMX/ETK, RLK/TXK and/or ERBB4 compared to BTK, ITKand/or compared to the others kinase from TEC, Etk, Txk and/or ErbB4,e.g. the inhibitor exhibits at least 1.5-fold, 2.5-fold, 5.0-fold,10.0-fold, 25-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold,500-fold, 1000-fold or more activity for one of TEC, BMX/ETK, RLK/TXKand/or ERBB4 compared to BTK, ITK and/or compared to another kinase fromTEC, BMX/ETK, RLK/TXK and/or ERBB4 that is distinct from the proteintyrosine kinase. In some embodiments, the cells and dosage regimens foradministering the inhibitor can include any as described in thefollowing subsection B under “Administration of Inhibitor.”

In some embodiments, the immunotherapy, such as a T cell therapy (e.g.CAR-expressing T cells) or a T cell-engaging therapy, and inhibitor areprovided as pharmaceutical compositions for administration to thesubject. In some embodiments, the pharmaceutical compositions containtherapeutically effective amounts of one or both of the agents forcombination therapy, e.g., T cells for adoptive cell therapy and aninhibitor as described. In some embodiments, the agents are formulatedfor administration in separate pharmaceutical compositions. In someembodiments, any of the pharmaceutical compositions provided herein canbe formulated in dosage forms appropriate for each route ofadministration.

In some embodiments, the combination therapy, which includesadministering the immunotherapy (e.g. T cell therapy, includingengineered cells, such as CAR-T cell therapy) and the inhibitor areadministered to a subject or patient having a disease or condition to betreated (e.g. cancer) or at risk for having the disease or condition(e.g. cancer). In some aspects, the methods treat, e.g., ameliorate oneor more symptom of, the disease or condition, such as by lessening tumorburden in a cancer expressing an antigen recognized by the immunotherapyor immunotherapeutic agent, e.g. recognized by an engineered T cell.

In some embodiments, the disease or condition that is treated can be anyin which expression of an antigen is associated with and/or involved inthe etiology of a disease condition or disorder, e.g. causes,exacerbates or otherwise is involved in such disease, condition, ordisorder. Exemplary diseases and conditions can include diseases orconditions associated with malignancy or transformation of cells (e.g.cancer), autoimmune or inflammatory disease, or an infectious disease,e.g. caused by bacterial, viral or other pathogens. Exemplary antigens,which include antigens associated with various diseases and conditionsthat can be treated, include any of antigens described herein. Inparticular embodiments, the recombinant receptor expressed on engineeredcells of a combination therapy, including a chimeric antigen receptor ortransgenic TCR, specifically binds to an antigen associated with thedisease or condition.

In some embodiments, the disease or condition is a tumor, such as asolid tumor, lymphoma, leukemia, blood tumor, metastatic tumor, or othercancer or tumor type.

In some embodiments, the cancer or proliferative disease is a B cellmalignancy or hematological malignancy. In some embodiments the canceror proliferative disease is lymphoblastic leukemia (ALL), non-Hodgkin'slymphoma (NHL), or chronic lymphocytic leukemia (CLL). In someembodiments, the cancer is CLL. In some embodiments, the methods can beused to treat a myeloma, a lymphoma or a leukemia. In some embodiments,the methods can be used to treat a non-Hodgkin lymphoma (NHL), an acutelymphoblastic leukemia (ALL), a chronic lymphocytic leukemia (CLL), adiffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), ora myeloma, e.g., a multiple myeloma (MM). In some embodiments, themethods can be used to treat a MM or a DBCBL.

In some embodiments, the antigen associated with the disease or disorderis selected from the group consisting of ROR1, B cell maturation antigen(BCMA), tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, andhepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30,CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbBdimers, EGFR vIII, FBP, FCRLS, FCRHS, fetal acethycholine receptor, GD2,GD3, BMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, LewisY, L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen(MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen ofmelanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptor a2(IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9,NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, dualantigen, and an antigen associated with a universal tag, a cancer-testesantigen, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1,gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen(CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor,progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2(OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138,and a pathogen-specific antigen. In some embodiments, the antigen isassociated with or is a universal tag.

In some embodiments the cancer or proliferative disease is not a cancerexpressing a B cell antigen. In some embodiments, the B cell antigen isselected from the group consisting of CD19, CD20, CD22 and ROR1. In someembodiments the cancer or proliferative disease is a non-hematologiccancer. In some embodiments the cancer or proliferative disease is asolid tumor. In some embodiments the cancer or proliferative diseasedoes not express CD19, CD20, CD22 or ROR1. In some embodiments, theprovided methods employ a recombinant receptor-expressing T cell (e.g.CAR-T cell) that does not target or specifically bind CD19, CD20, CD22or ROR1.

In some embodiments, the methods can be used to treat a non-hematologiccancer, such as a solid tumor. In some embodiments, the methods can beused to treat a bladder, lung, brain, melanoma (e.g. small-cell lung,melanoma), breast, cervical, ovarian, colorectal, pancreatic,endometrial, esophageal, kidney, liver, prostate, skin, thyroid, oruterine cancers. In some embodiments, the cancer or proliferativedisease is cancer is a pancreatic cancer, bladder cancer, colorectalcancer, breast cancer, prostate cancer, renal cancer, hepatocellularcancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer,rectal cancer, thyroid cancer, uterine cancer, gastric cancer,esophageal cancer, head and neck cancer, melanoma, neuroendocrinecancers, CNS cancers, brain tumors, bone cancer, or soft tissue sarcoma.

In some embodiments, the disease or condition is an infectious diseaseor condition, such as, but not limited to, viral, retroviral, bacterial,and protozoal infections, immunodeficiency, Cytomegalovirus (CMV),Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In someembodiments, the disease or condition is an autoimmune or inflammatorydisease or condition, such as arthritis, e.g., rheumatoid arthritis(RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatorybowel disease, psoriasis, scleroderma, autoimmune thyroid disease,Graves disease, Crohn's disease, multiple sclerosis, asthma, and/or adisease or condition associated with transplant.

For the prevention or treatment of disease, the appropriate dosage ofthe inhibitor, such as selective inhibitor, of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, and/or immunotherapy, such as a T celltherapy (e.g. CAR-expressing T cells) or a T cell-engaging therapy, maydepend on the type of disease to be treated, the particular inhibitor,cells and/or recombinant receptors expressed on the cells, the severityand course of the disease, route of administration, whether theinhibitor and/or the immunotherapy, e.g., T cell therapy, areadministered for preventive or therapeutic purposes, previous therapy,frequency of administration, the subject's clinical history and responseto the cells, and the discretion of the attending physician. Thecompositions and cells are in some embodiments suitably administered tothe subject at one time or over a series of treatments. Exemplary dosageregimens and schedules for the provided combination therapy aredescribed.

In some embodiments, the immunotherapy, e.g. T cell therapy, and theinhibitor, such as selective inhibitor, of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 are administered as part of a furthercombination treatment, which can be administered simultaneously with orsequentially to, in any order, another therapeutic intervention. In somecontexts, the immunotherapy, e.g. engineered T cells, such asCAR-expressing T cells, are co-administered with another therapysufficiently close in time such that the immunotherapy enhances theeffect of one or more additional therapeutic agents, or vice versa. Insome embodiments, the cells are administered prior to the one or moreadditional therapeutic agents. In some embodiments, the immunotherapy,e.g. engineered T cells, such as CAR-expressing T cells, areadministered after the one or more additional therapeutic agents. Insome embodiments, the combination therapy methods further include alymphodepleting therapy, such as administration of a chemotherapeuticagent. In some embodiments, the combination therapy further comprisesadministering another therapeutic agent, such as an anti-cancer agent, acheckpoint inhibitor, or another immune modulating agent. Uses includeuses of the combination therapies in such methods and treatments, anduses of such compositions in the preparation of a medicament in order tocarry out such combination therapy methods. In some embodiments, themethods and uses thereby treat the disease or condition or disorder,such as a cancer or proliferative disease, in the subject.

Prior to, during or following administration of the immunotherapy (e.g.T cell therapy, such as CAR-T cell therapy) and/or an inhibitor, such asselective inhibitor, of one or more of BTK, TEC, BMX/ETK, RLK/TXK and/orERBB4, the biological activity of the immunotherapy, e.g. the biologicalactivity of the engineered cell populations, in some embodiments ismeasured, e.g., by any of a number of known methods. Parameters toassess include the ability of the engineered cells to destroy targetcells, persistence and other measures of T cell activity, such asmeasured using any suitable method known in the art, such as assaysdescribed further below in Section IV below. In some embodiments, thebiological activity of the cells, e.g., T cells administered for the Tcell based therapy, is measured by assaying cytotoxic cell killing,expression and/or secretion of one or more cytokines, proliferation orexpansion, such as upon restimulation with antigen. In some aspects thebiological activity is measured by assessing the disease burden and/orclinical outcome, such as reduction in tumor burden or load. In someembodiments, administration of one or both agents of the combinationtherapy and/or any repeated administration of the therapy, can bedetermined based on the results of the assays before, during, during thecourse of or after administration of one or both agents of thecombination therapy.

In some embodiments, the combined effect of the inhibitor in combinationwith the cell therapy can be synergistic compared to treatmentsinvolving only the inhibitor or monotherapy with the cell therapy. Forexample, in some embodiments, the methods provided herein result in anincrease or an improvement in a desired therapeutic effect, such as anincreased or an improvement in the reduction or inhibition of one ormore symptoms associated with cancer.

In some embodiments, the inhibitor increases the expansion orproliferation of the engineered T cells, such as CAR T-Cells. In someembodiments, the increase in expansion or proliferation is observed invivo upon administration to a subject. In some embodiments, the increasein the number of engineered T cells, e.g. CAR-T cells, is increased bygreater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold,3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold,10.0 fold or more.

A. Administration of Immunotherapy (e.g. T Cell Therapy or TCell-Engaging Therapy)

In some embodiments of the methods, compositions, combinations, kits anduses provided herein, the combination therapy includes administering toa subject an immunotherapy, such as a T cell therapy (e.g.CAR-expressing T cells) or a T cell-engaging therapy. Such therapies canbe administered prior to, subsequent to, simultaneously withadministration of one or more inhibitor of a TEK family kinase asdescribed.

In some embodiments, the immunotherapy is a cell-based therapy that isor comprises administration of cells, such as immune cells, for exampleT cell or NK cells, that target a molecule expressed on the surface of alesion, such as a tumor or a cancer. In some embodiments, the immunecells express a T cell receptor (TCR) or other antigen-binding receptor.In some embodiments, the immune cells express a recombinant receptor,such as a transgenic TCR or a chimeric antigen receptor (CAR). In someembodiments, the cells are autologous to the subject. In someembodiments, the cells are allogeneic to the subject. Exemplary of suchcell therapies, e.g. T cell therapies, for use in the provided methodsare described below.

1. T Cell-Engaging Therapy

In some embodiments, the immunotherapy is or comprises a T cell-engagingtherapy that is or comprises a binding molecule capable of binding to asurface molecule expressed on a T cell. In some embodiments, the surfacemolecule is an activating component of a T cell, such as a component ofthe T cell receptor complex. In some embodiments, the surface moleculeis CD3 or is CD2. In some embodiments, the T cell-engaging therapy is orcomprises an antibody or antigen-binding fragment. In some embodiments,the T cell-engaging therapy is a bispecific antibody containing at leastone antigen-binding domain binding to an activating component of the Tcell (e.g. a T cell surface molecule, e.g. CD3 or CD2) and at least oneantigen-binding domain binding to a surface antigen on a target cell,such as a surface antigen on a tumor or cancer cell, for example any ofthe listed antigens as described herein, e.g. CD19. In some embodiments,the simultaneous or near simultaneous binding of such an antibody toboth of its targets can result in a temporary interaction between thetarget cell and T cell, thereby resulting in activation, e.g. cytotoxicactivity, of the T cell and subsequent lysis of the target cell.

Among such exemplary bispecific antibody T cell-engagers are bispecificT cell engager (BiTE) molecules, which contain tandem scFv moleculesfused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res317, 1255-1260 (2011); tandem scFv molecules fused to each other via,e.g. a flexible linker, and that further contain an Fc domain composedof a first and a second subunit capable of stable association(WO2013026837); diabodies and derivatives thereof, including tandemdiabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al,J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART)molecules that can include the diabody format with a C-terminaldisulfide bridge; or triomabs that include whole hybrid mouse/rat IgGmolecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010). In someembodiments, the T-cell engaging therapy is blinatumomab or AMG 330. Anyof such T cell-engagers can be used in used in the provided methods.

2. T Cell Therapy

In some aspects, the T cell therapy is or comprises a tumor infiltratinglymphocytic (TIL) therapy, a transgenic TCR therapy or a T cell therapycomprising genetically engineered cells, such as a recombinant-receptorexpressing cell therapy. In some embodiments, the recombinant receptorspecifically binds to a ligand, such as one associated with a disease orcondition, e.g. associated with or expressed on a cell of a tumor orcancer. In some embodiments, the T cell therapy includes administering Tcells engineered to express a chimeric antigen receptor (CAR).

In some embodiments, the provided cells express and/or are engineered toexpress receptors, such as recombinant receptors, including thosecontaining ligand-binding domains or binding fragments thereof, and Tcell receptors (TCRs) and components thereof, and/or functional non-TCRantigen receptors, such as chimeric antigen receptors (CARs). In someembodiments, the recombinant receptor contains an extracellularligand-binding domain that specifically binds to an antigen. In someembodiments, the recombinant receptor is a CAR that contains anextracellular antigen-recognition domain that specifically binds to anantigen. In some embodiments, the ligand, such as an antigen, is aprotein expressed on the surface of cells. In some embodiments, the CARis a TCR-like CAR and the antigen is a processed peptide antigen, suchas a peptide antigen of an intracellular protein, which, like a TCR, isrecognized on the cell surface in the context of a majorhistocompatibility complex (MEW) molecule.

Among the engineered cells, including engineered cells containingrecombinant receptors, are described in Section III below. Exemplaryrecombinant receptors, including CARs and recombinant TCRs, as well asmethods for engineering and introducing the receptors into cells,include those described, for example, in international patentapplication publication numbers WO200014257, WO2013126726,WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061U.S. patent application publication numbers US2002131960, US2013287748,US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592,8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209,7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patentapplication number EP2537416, and/or those described by Sadelain et al.,Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5):633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75. In some aspects, thegenetically engineered antigen receptors include a CAR as described inU.S. Pat. No. 7,446,190, and those described in International PatentApplication Publication No.: WO/2014055668 A1.

Methods for administration of engineered cells for adoptive cell therapyare known and may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.,(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al., (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al., (2013) PLoS ONE 8(4):e61338.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by autologous transfer, in which the cells are isolatedand/or otherwise prepared from the subject who is to receive the celltherapy, or from a sample derived from such a subject. Thus, in someaspects, the cells are derived from a subject, e.g., patient, in need ofa treatment and the cells, following isolation and processing areadministered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

The cells can be administered by any suitable means. The cells areadministered in a dosing regimen to achieve a therapeutic effect, suchas a reduction in tumor burden. Dosing and administration may depend inpart on the schedule of administration of the inhibitor of a TEC familykinase, which can be administered prior to, subsequent to and/orsimultaneously with initiation of administration of the T cell therapy.Various dosing schedules of the T cell therapy include but are notlimited to single or multiple administrations over various time-points,bolus administration, and pulse infusion.

a. Compositions and Formulations

In some embodiments, the dose of cells of the T cell therapy, such a Tcell therapy comprising cells engineered with a recombinant antigenreceptor, e.g. CAR or TCR, is provided as a composition or formulation,such as a pharmaceutical composition or formulation. Such compositionscan be used in accord with the provided methods, such as in theprevention or treatment of diseases, conditions, and disorders.

In some embodiments, the T cell therapy, such as engineered T cells(e.g. CAR T cells), are formulated with a pharmaceutically acceptablecarrier. In some aspects, the choice of carrier is determined in part bythe particular cell or agent and/or by the method of administration.Accordingly, there are a variety of suitable formulations. For example,the pharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition. Carriers are described, e.g., byRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being prevented ortreated with the cells or agents, where the respective activities do notadversely affect one another. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended. Thus, in some embodiments, the pharmaceutical compositionfurther includes other pharmaceutically active agents or drugs, such aschemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,vincristine, etc.

The pharmaceutical composition in some embodiments contains cells inamounts effective to treat or prevent the disease or condition, such asa therapeutically effective or prophylactically effective amount.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and can be determined. Thedesired dosage can be delivered by a single bolus administration of thecomposition, by multiple bolus administrations of the composition, or bycontinuous infusion administration of the composition.

The cells may be administered using standard administration techniques,formulations, and/or devices. Provided are formulations and devices,such as syringes and vials, for storage and administration of thecompositions. With respect to cells, administration can be autologous orheterologous. For example, immunoresponsive cells or progenitors can beobtained from one subject, and administered to the same subject or adifferent, compatible subject. Peripheral blood derived immunoresponsivecells or their progeny (e.g., in vivo, ex vivo or in vitro derived) canbe administered via localized injection, including catheteradministration, systemic injection, localized injection, intravenousinjection, or parenteral administration. When administering atherapeutic composition (e.g., a pharmaceutical composition containing agenetically modified immunoresponsive cell), it will generally beformulated in a unit dosage injectable form (solution, suspension,emulsion).

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, theagent or cell populations are administered parenterally. The term“parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. Insome embodiments, the agent or cell populations are administered to asubject using peripheral systemic delivery by intravenous,intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in some aspects bebuffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsin a solvent, such as in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can also be lyophilized. Thecompositions can contain auxiliary substances such as wetting,dispersing, or emulsifying agents (e.g., methylcellulose), pH bufferingagents, gelling or viscosity enhancing additives, preservatives,flavoring agents, colors, and the like, depending upon the route ofadministration and the preparation desired. Standard texts may in someaspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of agent oragents, the type of cells or recombinant receptors, the severity andcourse of the disease, whether the agent or cells are administered forpreventive or therapeutic purposes, previous therapy, the subject'sclinical history and response to the agent or the cells, and thediscretion of the attending physician. The compositions are in someembodiments suitably administered to the subject at one time or over aseries of treatments.

In some cases, the cell therapy is administered as a singlepharmaceutical composition comprising the cells. In some embodiments, agiven dose is administered by a single bolus administration of the cellsor agent. In some embodiments, it is administered by multiple bolusadministrations of the cells or agent, for example, over a period of nomore than 3 days, or by continuous infusion administration of the cellsor agent.

b. Dosage Schedule and Administration

In some embodiments, a dose of cells is administered to subjects inaccord with the provided methods. In some embodiments, the size ortiming of the doses is determined as a function of the particulardisease or condition in the subject. It is within the level of a skilledartisan to empirically determine the size or timing of the doses for aparticular disease in view of the provided description.

In certain embodiments, the cells, or individual populations ofsub-types of cells, are administered to the subject at a range of about0.1 million to about 100 billion cells and/or that amount of cells perkilogram of body weight of the subject, such as, e.g., 0.1 million toabout 50 billion cells (e.g., about 5 million cells, about 25 millioncells, about 500 million cells, about 1 billion cells, about 5 billioncells, about 20 billion cells, about 30 billion cells, about 40 billioncells, or a range defined by any two of the foregoing values), 1 millionto about 50 billion cells (e.g., about 5 million cells, about 25 millioncells, about 500 million cells, about 1 billion cells, about 5 billioncells, about 20 billion cells, about 30 billion cells, about 40 billioncells, or a range defined by any two of the foregoing values), such asabout 10 million to about 100 billion cells (e.g., about 20 millioncells, about 30 million cells, about 40 million cells, about 60 millioncells, about 70 million cells, about 80 million cells, about 90 millioncells, about 10 billion cells, about 25 billion cells, about 50 billioncells, about 75 billion cells, about 90 billion cells, or a rangedefined by any two of the foregoing values), and in some cases about 100million cells to about 50 billion cells (e.g., about 120 million cells,about 250 million cells, about 350 million cells, about 450 millioncells, about 650 million cells, about 800 million cells, about 900million cells, about 3 billion cells, about 30 billion cells, about 45billion cells) or any value in between these ranges and/or per kilogramof body weight of the subject. Dosages may vary depending on attributesparticular to the disease or disorder and/or patient and/or othertreatments. In some embodiments, such values refer to numbers ofrecombinant receptor-expressing cells; in other embodiments, they referto number of T cells or PBMCs or total cells administered.

In some embodiments, the cell therapy comprises administration of a dosecomprising a number of cells that is at least or at least about or is oris about 0.1×10⁶ cells/kg body weight of the subject, 0.2×10⁶ cells/kg,0.3×10⁶ cells/kg, 0.4×10⁶ cells/kg, 0.5×10⁶ cells/kg, 1×10⁶ cell/kg,2.0×10⁶ cells/kg, 3×10⁶ cells/kg or 5×10⁶ cells/kg.

In some embodiments, the cell therapy comprises administration of a dosecomprising a number of cells is between or between about 0.1×10⁶cells/kg body weight of the subject and 1.0×10⁷ cells/kg, between orbetween about 0.5×10⁶ cells/kg and 5×10⁶ cells/kg, between or betweenabout 0.5×10⁶ cells/kg and 3×10⁶ cells/kg, between or between about0.5×10⁶ cells/kg and 2×10⁶ cells/kg, between or between about 0.5×10⁶cells/kg and 1×10⁶ cell/kg, between or between about 1.0×10⁶ cells/kgbody weight of the subject and 5×10⁶ cells/kg, between or between about1.0×10⁶ cells/kg and 3×10⁶ cells/kg, between or between about 1.0×10⁶cells/kg and 2×10⁶ cells/kg, between or between about 2.0×10⁶ cells/kgbody weight of the subject and 5×10⁶ cells/kg, between or between about2.0×10⁶ cells/kg and 3×10⁶ cells/kg, or between or between about 3.0×10⁶cells/kg body weight of the subject and 5×10⁶ cells/kg, each inclusive.

In some embodiments, the dose of cells comprises between at or about2×10⁵ of the cells/kg and at or about 2×10⁶ of the cells/kg, such asbetween at or about 4×10⁵ of the cells/kg and at or about 1×10⁶ of thecells/kg or between at or about 6×10⁵ of the cells/kg and at or about8×10⁵ of the cells/kg. In some embodiments, the dose of cells comprisesno more than 2×10⁵ of the cells (e.g. antigen-expressing, such asCAR-expressing cells) per kilogram body weight of the subject(cells/kg), such as no more than at or about 3×10⁵ cells/kg, no morethan at or about 4×10⁵ cells/kg, no more than at or about 5×10⁵cells/kg, no more than at or about 6×10⁵ cells/kg, no more than at orabout 7×10⁵ cells/kg, no more than at or about 8×10⁵ cells/kg, nor morethan at or about 9×10⁵ cells/kg, no more than at or about 1×10⁶cells/kg, or no more than at or about 2×10⁶ cells/kg. In someembodiments, the dose of cells comprises at least or at least about orat or about 2×10⁵ of the cells (e.g. antigen-expressing, such asCAR-expressing cells) per kilogram body weight of the subject(cells/kg), such as at least or at least about or at or about 3×10⁵cells/kg, at least or at least about or at or about 4×10⁵ cells/kg, atleast or at least about or at or about 5×10⁵ cells/kg, at least or atleast about or at or about 6×10⁵ cells/kg, at least or at least about orat or about 7×10⁵ cells/kg, at least or at least about or at or about8×10⁵ cells/kg, at least or at least about or at or about 9×10⁵cells/kg, at least or at least about or at or about 1×10⁶ cells/kg, orat least or at least about or at or about 2×10⁶ cells/kg.

In certain embodiments, the cells, or individual populations ofsub-types of cells, are administered to the subject at a range of aboutone million to about 100 billion cells and/or that amount of cells perkilogram of body weight, such as, e.g., 1 million to about 50 billioncells (e.g., about 5 million cells, about 25 million cells, about 500million cells, about 1 billion cells, about 5 billion cells, about 20billion cells, about 30 billion cells, about 40 billion cells, or arange defined by any two of the foregoing values), such as about 10million to about 100 billion cells (e.g., about 20 million cells, about30 million cells, about 40 million cells, about 60 million cells, about70 million cells, about 80 million cells, about 90 million cells, about10 billion cells, about 25 billion cells, about 50 billion cells, about75 billion cells, about 90 billion cells, or a range defined by any twoof the foregoing values), and in some cases about 100 million cells toabout 50 billion cells (e.g., about 120 million cells, about 250 millioncells, about 350 million cells, about 450 million cells, about 650million cells, about 800 million cells, about 900 million cells, about 3billion cells, about 30 billion cells, about 45 billion cells) or anyvalue in between these ranges and/or per kilogram of body weight.Dosages may vary depending on attributes particular to the disease ordisorder and/or patient and/or other treatments.

In some embodiments, the dose of cells is a flat dose of cells or fixeddose of cells such that the dose of cells is not tied to or based on thebody surface area or weight of a subject.

In some embodiments, for example, where the subject is a human, the doseincludes fewer than about 1×10⁸ total recombinant receptor (e.g.,CAR)-expressing cells, T cells, or peripheral blood mononuclear cells(PBMCs), e.g., in the range of about 1×10⁶ to 1×10⁸ such cells, such as2×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, or 1×10⁸ or total such cells, or the rangebetween any two of the foregoing values. In some embodiments, where thesubject is a human, the dose includes between about 1×10⁶ and 3×10⁸total recombinant receptor (e.g., CAR)-expressing cells, e.g., in therange of about 1×10⁷ to 2×10⁸ such cells, such as 1×10⁷, 5×10⁷, 1×10⁸ or1.5×10⁸ total such cells, or the range between any two of the foregoingvalues. In some embodiments, the patient is administered multiple doses,and each of the doses or the total dose can be within any of theforegoing values. In some embodiments, the dose of cells comprises theadministration of from or from about 1×10⁵ to 5×10⁸ total recombinantreceptor-expressing T cells or total T cells, 1×10⁵ to 1×10⁸ totalrecombinant receptor-expressing T cells or total T cells, from or fromabout 5×10⁵ to 1×10⁷ total recombinant receptor-expressing T cells ortotal T cells, or from or from about 1×10⁶ to 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, each inclusive.

In some embodiments, the T cells of the dose include CD4+ T cells, CD8+T cells or CD4+ and CD8+ T cells.

In some embodiments, for example, where the subject is human, the CD8+ Tcells of the dose, including in a dose including CD4+ and CD8+ T cells,includes between about 1×10⁶ and 1×10⁸ total recombinant receptor (e.g.,CAR)-expressing CD8+ cells, e.g., in the range of about 5×10⁶ to 1×10⁸such cells, such cells 1×10⁷, 2.5×10⁷, 5×10⁷, 7.5×10⁷ or 1×10⁸ totalsuch cells, or the range between any two of the foregoing values. Insome embodiments, the patient is administered multiple doses, and eachof the doses or the total dose can be within any of the foregoingvalues. In some embodiments, the dose of cells comprises theadministration of from or from about 1×10⁷ to 0.75×10⁸ total recombinantreceptor-expressing CD8+ T cells, 1×10⁷ to 2.5×10⁷ total recombinantreceptor-expressing CD8+ T cells, from or from about 1×10⁷ to 0.75×10⁸total recombinant receptor-expressing CD8+ T cells, each inclusive. Insome embodiments, the dose of cells comprises the administration of orabout 1×10⁷, 2.5×10⁷, 5×10⁷ 7.5×10⁷ or 1×10⁸ total recombinantreceptor-expressing CD8+ T cells.

In some embodiments, the dose of cells, e.g., recombinantreceptor-expressing T cells, is administered to the subject as a singledose or is administered only one time within a period of two weeks, onemonth, three months, six months, 1 year or more.

In the context of adoptive cell therapy, administration of a given“dose” of cells encompasses administration of the given amount or numberof cells as a single composition and/or single uninterruptedadministration, e.g., as a single injection or continuous infusion, andalso encompasses administration of the given amount or number of cellsas a split dose, provided in multiple individual compositions orinfusions, over a specified period of time, such as no more than 3 days.Thus, in some contexts, the dose is a single or continuousadministration of the specified number of cells, given or initiated at asingle point in time. In some contexts, however, the dose isadministered in multiple injections or infusions over a period of nomore than three days, such as once a day for three days or for two daysor by multiple infusions over a single day period.

Thus, in some aspects, the cells of the dose are administered in asingle pharmaceutical composition. In some embodiments, the cells of thedose are administered in a plurality of compositions, collectivelycontaining the cells of the dose.

In some embodiments, the term “split dose” refers to a dose that issplit so that it is administered over more than one day. This type ofdosing is encompassed by the present methods and is considered to be asingle dose. In some embodiments, the cells of a split dose areadministered in a plurality of compositions, collectively comprising thecells of the dose, over a period of no more than three days.

Thus, the dose of cells may be administered as a split dose, e.g. asplit dose administered over time. For example, in some embodiments, thedose may be administered to the subject over 2 days or over 3 days.Exemplary methods for split dosing include administering 25% of the doseon the first day and administering the remaining 75% of the dose on thesecond day. In other embodiments, 33% of the dose may be administered onthe first day and the remaining 67% administered on the second day. Insome aspects, 10% of the dose is administered on the first day, 30% ofthe dose is administered on the second day, and 60% of the dose isadministered on the third day. In some embodiments, the split dose isnot spread over more than 3 days.

In some embodiments, the dose of cells is generally large enough to beeffective in reducing disease burden.

In some embodiments, the cells are administered at a desired dosage,which in some aspects includes a desired dose or number of cells or celltype(s) and/or a desired ratio of cell types. Thus, the dosage of cellsin some embodiments is based on a total number of cells (or number perkg body weight) and a desired ratio of the individual populations orsub-types, such as the CD4+ to CD8+ ratio. In some embodiments, thedosage of cells is based on a desired total number (or number per kg ofbody weight) of cells in the individual populations or of individualcell types. In some embodiments, the dosage is based on a combination ofsuch features, such as a desired number of total cells, desired ratio,and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8⁺and CD4⁺ T cells, are administered at or within a tolerated differenceof a desired dose of total cells, such as a desired dose of T cells. Insome aspects, the desired dose is a desired number of cells or a desirednumber of cells per unit of body weight of the subject to whom the cellsare administered, e.g., cells/kg. In some aspects, the desired dose isat or above a minimum number of cells or minimum number of cells perunit of body weight. In some aspects, among the total cells,administered at the desired dose, the individual populations orsub-types are present at or near a desired output ratio (such as CD4⁺ toCD8⁺ ratio), e.g., within a certain tolerated difference or error ofsuch a ratio.

In some embodiments, the cells are administered at or within a tolerateddifference of a desired dose of one or more of the individualpopulations or sub-types of cells, such as a desired dose of CD4+ cellsand/or a desired dose of CD8+ cells. In some aspects, the desired doseis a desired number of cells of the sub-type or population, or a desirednumber of such cells per unit of body weight of the subject to whom thecells are administered, e.g., cells/kg. In some aspects, the desireddose is at or above a minimum number of cells of the population orsub-type, or minimum number of cells of the population or sub-type perunit of body weight.

Thus, in some embodiments, the dosage is based on a desired fixed doseof total cells and a desired ratio, and/or based on a desired fixed doseof one or more, e.g., each, of the individual sub-types orsub-populations. Thus, in some embodiments, the dosage is based on adesired fixed or minimum dose of T cells and a desired ratio of CD4⁺ toCD8⁺ cells, and/or is based on a desired fixed or minimum dose of CD4⁺and/or CD8⁺ cells.

In some embodiments, the cells are administered at or within a toleratedrange of a desired output ratio of multiple cell populations orsub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects,the desired ratio can be a specific ratio or can be a range of ratios.for example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺to CD8⁺ cells) is between at or about 5:1 and at or about 5:1 (orgreater than about 1:5 and less than about 5:1), or between at or about1:3 and at or about 3:1 (or greater than about 1:3 and less than about3:1), such as between at or about 2:1 and at or about 1:5 (or greaterthan about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1,4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In someaspects, the tolerated difference is within about 1%, about 2%, about3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50% of the desired ratio,including any value in between these ranges.

In particular embodiments, the numbers and/or concentrations of cellsrefer to the number of recombinant receptor (e.g., CAR)-expressingcells. In other embodiments, the numbers and/or concentrations of cellsrefer to the number or concentration of all cells, T cells, orperipheral blood mononuclear cells (PBMCs) administered.

In some aspects, the size of the dose is determined based on one or morecriteria such as response of the subject to prior treatment, e.g.chemotherapy, disease burden in the subject, such as tumor load, bulk,size, or degree, extent, or type of metastasis, stage, and/or likelihoodor incidence of the subject developing toxic outcomes, e.g., CRS,macrophage activation syndrome, tumor lysis syndrome, neurotoxicity,and/or a host immune response against the cells and/or recombinantreceptors being administered.

In some embodiments, administration of the inhibitor in combination withthe cells is able to increase, in some cases significantly increase, theexpansion or proliferation of the cells, and thus a lower dose of cellscan be administered to the subject. In some cases, the provided methodsallow a lower dose of such cells to be administered, to achieve the sameor better efficacy of treatment as the dose in a method in which thecell therapy is administered without administering the inhibitor, suchas at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold lessthan the dose in a method in which the cell therapy is administeredwithout administering the inhibitor.

In some embodiments, for example, the lower dose contains less thanabout 5×10⁶ cells, recombinant receptor (e.g. CAR)-expressing cells, Tcells, and/or PBMCs per kilogram body weight of the subject, such asless than about 4.5×10⁶, 4×10⁶, 3.5×10⁶, 3×10⁶, 2.5×10⁶, 2×10⁶, 1.5×10⁶,1×10⁶, 5×10⁵, 2.5×10⁵, or 1×10⁵ such cells per kilogram body weight ofthe subject. In some embodiments, the lower dose contains less thanabout 1×10⁵, 2×10⁵, 5×10⁵, or 1×10⁶ of such cells per kilogram bodyweight of the subject, or a value within the range between any two ofthe foregoing values. In some embodiments, such values refer to numbersof recombinant receptor-expressing cells; in other embodiments, theyrefer to number of T cells or PBMCs or total cells administered.

In some embodiments, one or more subsequent dose of cells can beadministered to the subject. In some embodiments, the subsequent dose ofcells is administered greater than or greater than about 7 days, 14days, 21 days, 28 days or 35 days after initiation of administration ofthe first dose of cells. The subsequent dose of cells can be more than,approximately the same as, or less than the first dose. In someembodiments, administration of the T cell therapy, such asadministration of the first and/or second dose of cells, can berepeated.

In some embodiments, initiation of administration of the cell therapy,e.g. the dose of cells or a first dose of a split dose of cells, isadministered before (prior to), concurrently with or after (subsequentlyor subsequent to) the administration of the inhibitor.

In some embodiments, the dose of cells, or the subsequent dose of cells,is administered concurrently with or after starting or initiatingadministration of the inhibitor o. In some embodiments, the dose ofcells, or the subsequent dose of cells, is administered 0 to 90 days,such as 0 to 30 days, 0 to 15 days, 0 to 6 days, 0 to 96 hours, 0 to 24hours, 0 to 12 hours, to 6 hours, or 0 to 2 hours, 2 hours to 30 days, 2hours to 15 days, 2 hours to 6 days, 2 hours to 96 hours, 2 hours to 24hours, 2 hours to 12 hours, 2 hours to 6 hours, 6 hours to 90 days, 6hours to 30 days, 6 hours to 15 days, 6 hours to 6 days, 6 hours to 96hours, 6 hours to 24 hours, 6 hours to 12 hours, 12 hours to 90 days, 12hours to 30 days, 12 hours to 15 days, 12 hours to 6 days, 12 hours to96 hours, 12 hours to 24 hours, 24 hours to 90 days, 24 hours to 30days, 24 hours to 15 days, 24 hours to 6 days, 24 hours to 96 hours, 96hours to 90 days, 96 hours to 30 days, 96 hours to 15 days, 96 hours to6 days, 6 days to 90 days, 6 days to 30 days, 6 days to 15 days, 15 daysto 90 days, 15 days to 30 days or 30 days to 90 days after starting orinitiating administration of the inhibitor. In some embodiments, thedose of cells is administered at least or about at least or about 1hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 6 days, 12days, 15 days, 30 days, 60 days or 90 days after starting or initiatingadministration of the inhibitor.

In some embodiments, the dose of cells is administered at a time whenone or more effects of the inhibitor are achieved.

In some embodiments, the dose of cells, or the subsequent dose of cells,is administered prior to starting or initiating administration of theinhibitor. In some embodiments, the dose of cells is administered atleast or at least about 1 hour, at least or at least about 2 hours, atleast or at least about 3 hours, at least or at least about 6 hours, atleast or at least about 12 hours, at least or at least about 1 day, atleast or at least about 2 days, at least or at least about 3 days, atleast or about at least 4 days, at least or at least about 5 days, atleast or about at least 6 days, at least or at least about 7 days, atleast or about at least 12 days, at least or at least about 14 days, atleast or about at least 15 days, at least or at least about 21 days, atleast or at least about 28 days, at least or about at least 30 days, atleast or at least about 35 days, at least or at least about 42 days, atleast or about at least 60 days or at least or about at least 90 daysprior to administering the inhibitor.

In some embodiments, the administration of the inhibitor is at a time inwhich the prior administration of the immunotherapy (e.g. T celltherapy, such as CAR-T cell therapy) is associated with, or is likely tobe associated with, a decreased functionality of the T cells compared tothe functionality of the T cells at a time just prior to initiation ofthe immunotherapy (e.g. T cell therapy, such as CAR-T cell therapy) orat a preceding time point after initiation of the immunotherapy. In someembodiments, the method involves, subsequent to administering the doseof cells of the T cell therapy, e.g., adoptive T cell therapy, but priorto administering the inhibitor, assessing a sample from the subject forone or more function of T cells, such as expansion or persistence of thecells, e.g. as determined by level or amount in the blood, or otherphenotypes or desired outcomes as described herein, e.g., such as thosedescribed in Section III. Various parameters for determining orassessing the regimen of the combination therapy are described inSection III.

B. Administration of Inhibitor

The provided methods, compositions, combinations, kits and uses involveadministration of an inhibitor of a protein-tyrosine kinase selectedfrom Bruton's tyrosine kinase (Btk), tec protein tyrosine kinase (TEC),BMX non-receptor tyrosine kinase (BMX/ETK), TXK tyrosine kinase (TXK;RLK/TXK) and/or receptor tyrosine-protein kinase ErbB4 (ERBB4). Theinhibitor can be administered prior to, subsequently to, during,simultaneously or near simultaneously, sequentially and/orintermittently with administration of the immunotherapeutic agent orimmunotherapy, e.g., T cell therapy, e.g., administration of T cellsexpressing a chimeric antigen receptor (CAR).

In some embodiments, the inhibitor inhibits BTK with a half-maximalinhibitory concentration (IC50) of less than or less than about 1000 nM,less than or less than about 900 nM, less than or less than about 800nM, less than or less than about 700 nM, less than or less than about600 nM, less than or less than about 500 nM, less than or less thanabout 400 nM, less than or less than about 300 nM, less than or lessthan about 200 nM, or less than or less than about 100 nM. In someembodiments, the inhibitor binds to BTK with a dissociation constant(Kd) of less than or less than about 1000 nM, less than or less thanabout 900 nM, less than or less than about 800 nM, less than or lessthan about 700 nM, less than or less than about 600 nM, less than orless than about 500 nM, less than or less than about 400 nM, less thanor less than about 300 nM, less than or less than about 200 nM, or lessthan or less than about 100 nM. In some embodiments, the inhibitionconstant (Ki) of the inhibitor for BTK is less than or less than about1000 nM, less than or less than about 900 nM, less than or less thanabout 800 nM, less than or less than about 700 nM, less than or lessthan about 600 nM, less than or less than about 500 nM, less than orless than about 400 nM, less than or less than about 300 nM, less thanor less than about 200 nM, or less than or less than about 100 nM.

In some embodiments, the inhibitor inhibits TEC with a half-maximalinhibitory concentration (IC₅₀) of less than or less than about 1000 nM,less than or less than about 900 nM, less than or less than about 800nM, less than or less than about 700 nM, less than or less than about600 nM, less than or less than about 500 nM, less than or less thanabout 400 nM, less than or less than about 300 nM, less than or lessthan about 200 nM, or less than or less than about 100 nM. In someembodiments, the inhibitor binds to TEC with a dissociation constant(Kd) of less than or less than about 1000 nM, less than or less thanabout 900 nM, less than or less than about 800 nM, less than or lessthan about 700 nM, less than or less than about 600 nM, less than orless than about 500 nM, less than or less than about 400 nM, less thanor less than about 300 nM, less than or less than about 200 nM, or lessthan or less than about 100 nM. In some embodiments, the inhibitionconstant (Ki) of the inhibitor for TEC is less than or less than about1000 nM, less than or less than about 900 nM, less than or less thanabout 800 nM, less than or less than about 700 nM, less than or lessthan about 600 nM, less than or less than about 500 nM, less than orless than about 400 nM, less than or less than about 300 nM, less thanor less than about 200 nM, or less than or less than about 100 nM.

In some embodiments, the inhibitor inhibits BMX/ETK with a half-maximalinhibitory concentration (IC₅₀) of less than or less than about 1000 nM,less than or less than about 900 nM, less than or less than about 800nM, less than or less than about 700 nM, less than or less than about600 nM, less than or less than about 500 nM, less than or less thanabout 400 nM, less than or less than about 300 nM, less than or lessthan about 200 nM, or less than or less than about 100 nM. In someembodiments, the inhibitor binds to BMX/ETK with a dissociation constant(Kd) of less than or less than about 1000 nM, less than or less thanabout 900 nM, less than or less than about 800 nM, less than or lessthan about 700 nM, less than or less than about 600 nM, less than orless than about 500 nM, less than or less than about 400 nM, less thanor less than about 300 nM, less than or less than about 200 nM, or lessthan or less than about 100 nM. In some embodiments, the inhibitionconstant (Ki) of the inhibitor for BMX/ETK is less than or less thanabout 1000 nM, less than or less than about 900 nM, less than or lessthan about 800 nM, less than or less than about 700 nM, less than orless than about 600 nM, less than or less than about 500 nM, less thanor less than about 400 nM, less than or less than about 300 nM, lessthan or less than about 200 nM, or less than or less than about 100 nM.

In some embodiments, the inhibitor inhibits RLK/TXK with a half-maximalinhibitory concentration (IC₅₀) of less than or less than about 1000 nM,less than or less than about 900 nM, less than or less than about 800nM, less than or less than about 700 nM, less than or less than about600 nM, less than or less than about 500 nM, less than or less thanabout 400 nM, less than or less than about 300 nM, less than or lessthan about 200 nM, or less than or less than about 100 nM. In someembodiments, the inhibitor binds to RLK/TXK with a dissociation constant(Kd) of less than or less than about 1000 nM, less than or less thanabout 900 nM, less than or less than about 800 nM, less than or lessthan about 700 nM, less than or less than about 600 nM, less than orless than about 500 nM, less than or less than about 400 nM, less thanor less than about 300 nM, less than or less than about 200 nM, or lessthan or less than about 100 nM. In some embodiments, the inhibitionconstant (Ki) of the inhibitor for RLK/TXK is less than or less thanabout 1000 nM, less than or less than about 900 nM, less than or lessthan about 800 nM, less than or less than about 700 nM, less than orless than about 600 nM, less than or less than about 500 nM, less thanor less than about 400 nM, less than or less than about 300 nM, lessthan or less than about 200 nM, or less than or less than about 100 nM.

In some embodiments, the inhibitor inhibits ERBB4 with a half-maximalinhibitory concentration (IC₅₀) of less than or less than about 1000 nM,less than or less than about 900 nM, less than or less than about 800nM, less than or less than about 700 nM, less than or less than about600 nM, less than or less than about 500 nM, less than or less thanabout 400 nM, less than or less than about 300 nM, less than or lessthan about 200 nM, or less than or less than about 100 nM. In someembodiments, the inhibitor binds to ERBB4 with a dissociation constant(Kd) of less than or less than about 1000 nM, less than or less thanabout 900 nM, less than or less than about 800 nM, less than or lessthan about 700 nM, less than or less than about 600 nM, less than orless than about 500 nM, less than or less than about 400 nM, less thanor less than about 300 nM, less than or less than about 200 nM, or lessthan or less than about 100 nM. In some embodiments, the inhibitionconstant (Ki) of the inhibitor for ERBB4 is less than or less than about1000 nM, less than or less than about 900 nM, less than or less thanabout 800 nM, less than or less than about 700 nM, less than or lessthan about 600 nM, less than or less than about 500 nM, less than orless than about 400 nM, less than or less than about 300 nM, less thanor less than about 200 nM, or less than or less than about 100 nM.

In some embodiments, the inhibitor does not inhibit IL-2 inducibleT-cell kinase (ITK). In some embodiments, the inhibitor exhibits ahalf-maximal inhibitory concentration (IC₅₀) for ITK of greater than orgreater than about 1000 nM, greater than or greater than about 2 μM,greater than or greater than about 3 μM, greater than or greater thanabout 4 μM, greater than or greater than about 5 μM, greater than orgreater than about 10 μM, greater than or greater than about 50 μM,greater than or greater than about 100 μM, or greater than or greaterthan about 1000 μM. In some embodiments, the dissociation constant (Kd)of the inhibitor for ITK is greater than or greater than about 1000 nM,greater than or greater than about 2 μM, greater than or greater thanabout 3 μM, greater than or greater than about 4 μM, greater than orgreater than about 5 μM, greater than or greater than about 10 μM,greater than or greater than about 50 μM, greater than or greater thanabout 100 μM, or greater than or greater than about 1000 μM.

In some embodiments, the inhibitor inhibits one of more of BTK, TEC,BMX/ETK, RLK/TXK or ERBB4 with a half-maximal inhibitory concentration(IC₅₀) that is lower than that of ITK by at least about 2-fold, at leastabout 3-fold, at least about 4-fold, at least about 5-fold, at leastabout 10-fold, at least about 20-fold, at least about 30-fold, at leastabout 40-fold, at least about 50-fold, at least about 100-fold, at leastabout 500-fold, at least about 1000-fold, at least about 10,000-fold, atleast about 100,000-fold, or at least about 1,000,000-fold.

In some embodiments, the dissociation constant (Kd) of the inhibitor forone of more of BTK, TEC, BMX/ETK, RLK/TXK or ERBB4 is lower than that ofITK by at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 10-fold, at least about20-fold, at least about 30-fold, at least about 40-fold, at least about50-fold, at least about 100-fold, at least about 500-fold, at leastabout 1000-fold, at least about 10,000-fold, at least about100,000-fold, or at least about 1,000,000-fold.

In some embodiments, the inhibition constant (Ki) of the inhibitor forone of more of BTK, TEC, BMX/ETK, RLK/TXK or ERBB4 is lower than that ofITK by at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 10-fold, at least about20-fold, at least about 30-fold, at least about 40-fold, at least about50-fold, at least about 100-fold, at least about 500-fold, at leastabout 1000-fold, at least about 10,000-fold, at least about100,000-fold, or at least about 1,000,000-fold.

In some embodiments, the IC₅₀, Kd and/or Ki is measured or determinedusing an in vitro assay. Assays to assess or quantitate or measureactivity of protein tyrosine kinase inhibitors as described are known inthe art. Such assays can be conducted in vitro and include assays toassess the ability of an agent to inhibit a specific biological orbiochemical function. In some embodiments. In some embodiments, kinaseactivity studies can be performed. Protein tyrosine kinases catalyze thetransfer of the terminal phosphate group from adenosine triphosphate(ATP) to the hydroxyl group of a tyrosine residue of the kinase itselfor another protein substrate. In some embodiments, kinase activity canbe measured by incubating the kinase with the substrate (e.g.,inhibitor) in the presence of ATP. In some embodiments, measurement ofthe phosphorylated substrate by a specific kinase can be assessed byseveral reporter systems including colorimetric, radioactive, andfluorometric detection. (Johnson, S. A. & T. Hunter (2005) Nat. Methods2:17.) In some embodiments, inhibitors can be assessed for theiraffinity for a particular kinase or kinases, such as by usingcompetition ligand binding assays (Ma et al., Expert Opin Drug Discov.2008 June; 3(6):607-621) From these assays, the half-maximal inhibitoryconcentration (IC₅₀) can be calculated. IC₅₀ is the concentration thatreduces a biological or biochemical response or function by 50% of itsmaximum. In some cases, such as in kinase activity studies, IC₅₀ is theconcentration of the compound that is required to inhibit the targetkinase activity by 50%. In some cases, the dissociation constant (Kd)and/or the inhibition constant (Ki values) can be determinedadditionally or alternatively. IC₅₀ and Kd can be calculated by anynumber of means known in the art. The inhibition constant (Ki values)can be calculated from the IC₅₀ and Kd values according to theCheng-Prusoff equation: Ki=IC₅₀/(1+L/Kd), where L is the concentrationof the inhibitor (Biochem Pharmacol 22: 3099-3108, 1973). Ki is theconcentration of unlabeled inhibitor that would cause occupancy of 50%of the binding sites present in the absence of ligand or othercompetitors.

In some embodiments, the inhibitor is a peptide, protein, antibody, orantigen-binding fragment thereof, an antibody mimetic, an aptamer, or anucleic acid molecule. In some embodiments, the inhibitor is a smallmolecule.

In some embodiments, the inhibitor is an inhibitor of a tyrosine proteinkinase that has an accessible cysteine residue near the active site ofthe tyrosine kinase. In some embodiments, the inhibitor irreversiblyreduces or eliminates the activation of tyrosine kinase. In someembodiments, the inhibitor forms a covalent bond with a cysteine residueon the protein tyrosine kinase. In some embodiments, the cysteineresidue is a Cys 481 residue. In some embodiments, the inhibitorcomprises a Michael acceptor moiety that forms a covalent bond with theappropriate cysteine residue of the tyrosine kinase. In someembodiments, the Michael acceptor moiety preferentially binds with theappropriate cysteine side chain of the tyrosine kinase protein relativeto other biological molecules that also contain an assessable —SHmoiety.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of at or about 5.1 nM; does not exhibitinhibitory activity against Itk or exhibits any such activity with anIC₅₀ value of greater than at or about 1000 nM; exhibits inhibitoryactivity against Tec, e.g., with an IC₅₀ value of at or about 93 nM;exhibits inhibitory activity against RLK/TXK, e.g., with an IC₅₀ valueof at or about 368 nM; exhibits inhibitory activity against BMX/ETK withan IC₅₀ value of at or about 46 nM; and/or exhibits inhibitory activityagainst ErbB4, e.g., with an IC₅₀ value of at or about 16 nM, in eachcase optionally as measured by a known in vitro assay and/or assaydescribed herein. In some aspects, the inhibitor is Formula (II) or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Byrd J C, Harrington B, O'Brien S,Jones J A, Schuh A, Devereux S, et al. The compound of Formula (II) inrelapsed chronic lymphocytic leukemia. N Engl J Med. 2016;374(4):323-32; Wu et al., “The compound of Formula (II): a selectivesecond-generation BTK inhibitor,” Journal of Hematology & Oncology(2016) 9:21.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of at or about 1 μM, such as, for example,as measured by a known in vitro assay and/or assay described herein. Insome aspects, the inhibitor is BGB-3111 (described by Wu et al.,“Second-generation inhibitors of Bruton tyrosine kinase,” Journal ofHematology & Oncology (2016) 9:80 WO2016/024230), or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, polymorph or prodrugthereof. In some embodiments, the inhibitor exhibits inhibitory activityagainst Btk, e.g., with an IC₅₀ value of at or about 1.9 nM; does notexhibit inhibitory activity against Itk or exhibits any such activitywith an IC₅₀ value of greater than at or about 1000 nM or at or about4000 or 4270 nM, or has the same less inhibitory activity towards ITK asthe compound of Formula (II). In some embodiments, the inhibitor doesnot inhibit TEC or exhibits any such inhibitory activity against IC₅₀only with an IC₅₀ value greater than 1000 nM or greater than 10,000 nM.In some embodiments, the inhibitor does not inhibit BMX or exhibits anysuch inhibitory activity against IC₅₀ only with an IC₅₀ value greaterthan 1000 nM or at or about or greater than 1800 nM or greater than10,000 nM. In some aspects, the inhibitor is CGI-1746 (see Hendriks etal., “Targeting Bruton's tyrosine kinase in B cell malignancies,”Nature, 2014, 14: 219-232; Akinleye et al., “Ibrutinib and novel BTKinhibitors in clinical development.” Journal of Hematology & Oncology2013, 6:59; WO2016/024230; Di Paolo et al., Nat. Chem. Biol., 2011,7(1): 41-50), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of less than about 4.4 nM or about 5 nM;does not exhibit inhibitory activity against Itk or exhibits any suchactivity with an IC₅₀ value of greater than at or about 3000 nM;exhibits inhibitory activity against Tec, e.g., with an IC₅₀ value of ator about 8.2 nM or about 6.2 nM; exhibits inhibitory activity againstRLK/TXK, e.g., with an IC₅₀ value of at or about 1.9 nM or 1.4 nM;and/or exhibits inhibitory activity against BMX/ETK with an IC₅₀ valueof at or about 1.9 nM or 0.7 nM, in each case optionally as measured bya known in vitro assay and/or assay described herein. In some aspects,the inhibitor isN-(3-(2-(3-Aminophenylamino)pyrimidin-5-ylcarbamoyl)-4-methylphenyl)-2-naphthamidecompoundsorN-(2-(3-(2-Acrylamidoacetamido)phenylamino)pyrimidin-5-yl)-2-methyl-5-(3-(trifluoromethyl)benzamido)benzamide(Compounds 31 or 38) (Li et. al., J. Med. Chem., 2014, 57(12): 5112-28),or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof.

In some embodiments, the inhibitor exhibits inhibitory activity againstErbB4, e.g., with an IC₅₀ value of about 50 nM; and does not exhibitinhibitory activity against Itk or exhibits any such activity with anIC₅₀ value of greater than at or about 10 in each case optionally asmeasured by a known in vitro assay and/or assay described herein. Insome aspects, the inhibitor is 4557W (CAS ID 179248-61-4), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some embodiments, the inhibitor exhibits inhibitory activity againstErbB4, e.g., with an IC₅₀ value of about 5 nM to about 500 nM; and doesnot exhibit inhibitory activity against Itk or exhibits any suchactivity with an IC₅₀ value of greater than at or about 10 in each caseoptionally as measured by a known in vitro assay and/or assay describedherein. In some embodiments, the inhibitor exhibits inhibitory activityagainst ErbB4, e.g., with an IC₅₀ value of about 6.3 nM. In someaspects, the inhibitor is Afatinib (CAS ID 439081-18-2), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Davis, et al., Nat Biotechnol, 2011;29:1046-51. In some embodiments, the inhibitor exhibits inhibitoryactivity against ErbB4, e.g., with an IC₅₀ value of about 250 nM. Insome aspects, the inhibitor is AG1478 (CAS ID 175178-82-2) or Compound56 (CAS ID 171745-13-4), or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, polymorph or prodrug thereof. See Anastassiasdis etal., Nat Biotechnol. 2011 Oct. 30; 29(11): 1039-45. In some embodiments,the inhibitor exhibits inhibitory activity against ErbB4, e.g., with anIC₅₀ value of about 150 nM. In some aspects, the inhibitor is Gefitnib(CAS ID 184475-35-2), or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, polymorph or prodrug thereof. See Karaman et al.,Nat Biotechnol. 2008 January; 26(1): 127-32. In some aspects, theinhibitor is WHI-P154 (CAS ID 211555-04-3), or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, polymorph or prodrugthereof. See Anastassiasdis et al., Nat Biotechnol. 2011 Oct. 30;29(11): 1039-45. In some embodiments, the inhibitor exhibits inhibitoryactivity against ErbB4, e.g., with an IC₅₀ value of about 21 nM. In someaspects, the inhibitor is JNJ-28871063 (CAS ID 944341-54-2), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Emanuel et al., Mol Pharmacol. 2008February; 73(2):338-48. In some embodiments, the inhibitor exhibitsinhibitory activity against ErbB4, e.g., with an IC₅₀ value of about 18nM. In some aspects, the inhibitor is Kinome 714, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, polymorph or prodrugthereof. See Fidanze et al., Bioorg Med Chem Lett. 2010 Apr. 15;20(8):2452-5. In some embodiments, the inhibitor exhibits inhibitoryactivity against ErbB4, e.g., with a Kd value of about 21 nM. In someaspects, the inhibitor is Pelitinib (CAS ID 257933-82-7), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Fabian et al., Nat Biotechnol. 2005March; 23(3)329-36. In some embodiments, the inhibitor exhibitsinhibitory activity against ErbB4, e.g., with a Kd value of about 230nM. In some aspects, the inhibitor is Erlotinib (CAS ID 183319-69-9), ora pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Davis, et al., Nat Biotechnol, 2011;29:1046-51. In some embodiments, the inhibitor exhibits inhibitoryactivity against ErbB4, e.g., with an IC₅₀ value of about 345 nM. Insome aspects, the inhibitor is Lapatinib (CAS ID 183319-69-9), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Mirams et al., J Pharmacol ToxicolMethods, 2014; 70:246-54.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of less than about 250 nM; does notexhibit inhibitory activity against Itk or exhibits any such activitywith an IC₅₀ value of greater than at or about 5 μM; and/or exhibitsinhibitory activity against RLK/TXK, e.g., with an IC₅₀ value of at orabout 250 nM, in each case optionally as measured by a known in vitroassay and/or assay described herein. In some aspects, the inhibitor isAloisine A (CAS ID 496864-16-5), or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, polymorph or prodrug thereof. SeeAnastassiasdis et al., Nat Biotechnol. 2011 Oct. 30; 29(11): 1039-45.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of about 545 nM; and does not exhibitinhibitory activity against Itk or exhibits any such activity with anIC₅₀ value of greater than at or about 25 in each case optionally asmeasured by a known in vitro assay and/or assay described herein. Insome aspects, the inhibitor is AMG-47a (CAS ID 882663-88-9), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See DiMauro et al., J Med Chem, 2006 Sep.21; 49(19): 5671-86.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of less than about 500 nM; does notexhibit inhibitory activity against Itk or exhibits any such activitywith an IC₅₀ value of greater than at or about 10 μM; and/or exhibitsinhibitory activity against RLK/TXK, e.g., with an IC₅₀ value of at orabout 500 nM, in each case optionally as measured by a known in vitroassay and/or assay described herein. In some aspects, the inhibitor isAS601245 (CAS ID 345987-15-7), or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, polymorph or prodrug thereof. SeeAnastassiasdis et al., Nat Biotechnol. 2011 Oct. 30; 29(11): 1039-45.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an Kd value of less than about 50 nM; does not exhibitinhibitory activity against Itk or exhibits any such activity with anIC₅₀ value of greater than at or about 5000 nM; exhibits inhibitoryactivity against RLK/TXK, e.g., with an Kd value of at or about 50 nM;and/or exhibits inhibitory activity against ErbB4 with an IC₅₀ value ofabout 60 nM, in each case optionally as measured by a known in vitroassay and/or assay described herein. In some aspects, the inhibitor isBMS-690514 (CAS ID 859853-30-8), or a pharmaceutically acceptable salt,solvate, hydrate, co-crystal, polymorph or prodrug thereof. See Wong, etal., Clin Cancer Res. 2011 Jun. 15; 17(12):4031-41.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of less than about 2.5 nM; does notexhibit inhibitory activity against Itk or exhibits any such activitywith an Kd value of greater than at or about 1700 nM; exhibitsinhibitory activity against Tec, e.g., with an IC₅₀ value of at or about282 nM; exhibits inhibitory activity against RLK/TXK, e.g., with an IC₅₀value of at or about 40 nM; exhibits inhibitory activity against BMX/ETKwith an IC₅₀ value of at or about 7.9 nM; and exhibits inhibitoryactivity against ErbB4 with an IC₅₀ value of at or about 2.5 nM, in eachcase optionally as measured by a known in vitro assay and/or assaydescribed herein. In some aspects, the inhibitor is Bosutinib (CAS ID380843-75-4), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof. See Remsing et al., Leukemia.2009 March; 23(3): 477-85.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of about 185 nM; does not exhibitinhibitory activity against Itk or exhibits any such activity with an Kdvalue of greater than at or about 5600 nM; exhibits inhibitory activityagainst RLK/TXK, e.g., with an Kd value of at or about 700 nM; exhibitsinhibitory activity against BMX/ETK with an IC₅₀ value of at or about 62nM; and exhibits inhibitory activity against ErbB4 with an Kd value ofat or about 6.5 nM, in each case optionally as measured by a known invitro assay and/or assay described herein. In some aspects, theinhibitor is Canertinib (CAS ID 289499-45-2), or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, polymorph or prodrugthereof. See Hur et al., Bioorg Med Chem Lett. 2008 Nov. 15;18(22):5916-9.

In some embodiments, the inhibitor exhibits inhibitory activity againstBtk, e.g., with an IC₅₀ value of about 2 nM; does not exhibit inhibitoryactivity against Itk or exhibits any such activity with an Kd value ofgreater than at or about 10000 nM; exhibits inhibitory activity againstRLK/TXK, e.g., with an IC₅₀ value of at or about 2 nM; and/or exhibitsinhibitory activity against BMX/ETK with an Kd value of at or about 36nM; in each case optionally as measured by a known in vitro assay and/orassay described herein. In some aspects, the inhibitor is CHEMBL249097,or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Bamborough et al., Bioorg Med ChemLett. 2007 Aug. 1; 17(15):4363-8.

In some aspects, the inhibitor is CHEMBL383899 (CAS ID 879127-16-9), ora pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is CP724714 (CAS ID 537705-08-1), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Karaman et al., Nat Biotechnol. 2008January; 26(1): 127-32.

In some aspects, the inhibitor is Dasatinib (CAS ID 302962-49-8), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Remsing et al., Leukemia. 2009 March;23(3): 477-85.

In some aspects, the inhibitor is GSK-3 Inhibitor X (CAS ID740841-15-0), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof. See Anastassiasdis et al., NatBiotechnol. 2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is HDS029 (CAS ID 881001-19-0), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is IKK-2 Inhibitor IV (CAS ID507475-17-4), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof.

In some aspects, the inhibitor is JNJ-10198409 (CAS ID 627518-40-5), ora pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is Ki11502 (CAS ID 347155-76-4), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is Lck Inhibitor (CAS ID 213743-31-8), ora pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is MK5108 (CAS ID 1010085-13-8), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Shimomura et al., Mol Cancer Ther.2010 January; 9(1):157-66.

In some aspects, the inhibitor is N-Benzoylstaurosporine (CAS ID120685-11-2), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof. See Anastassiasdis et al., NatBiotechnol. 2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is Neratinib (CAS ID 698387-09-6), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Davis, et al., Nat Biotechnol, 2011;29:1046-51.

In some aspects, the inhibitor is NU6140 (CAS ID 444723-13-1), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is Pazopanib (CAS ID 444731-52-6), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is PD168393 (CAS ID 194423-15-9), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Hur et al., Bioorg Med Chem Lett. 2008Nov. 15; 18(22):5916-9.

In some aspects, the inhibitor is PD169316 (CAS ID 152121-53-4), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is PD173955 (CAS ID 260415-63-2), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Davis, et al., Nat Biotechnol, 2011;29:1046-51.

In some aspects, the inhibitor is PDK1/Akt/Flt Dual Pathway Inhibitor(CAS ID 331253-86-2), or a pharmaceutically acceptable salt, solvate,hydrate, co-crystal, polymorph or prodrug thereof. See Anastassiasdis etal., Nat Biotechnol. 2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is Ponatinib (CAS ID 943319-70-8), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Huang et al., J Med Chem. 2010 Jun.24; 53(12):4701-19.

In some aspects, the inhibitor is PP1 Analog II; 1NM-PP1 (CAS ID221244-14-0), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof. See Anastassiasdis et al., NatBiotechnol. 2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is PP121 (CAS ID 1092788-83-4), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Apsel et al., Nat Chem Biol. 2008November; 4(11):691-9.

In some aspects, the inhibitor is Purvalanol A (CAS ID 212844-53-6), ora pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is Src Kinase Inhibitor I (CAS ID179248-59-0), or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof. See Anastassiasdis et al., NatBiotechnol. 2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is SureCN7018367, or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, polymorph or prodrugthereof. See Bamborough et al., J Med Chem. 2008 Dec. 25;51(24):7898-914.

In some aspects, the inhibitor is TWS119 (CAS ID 601514-19-6), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is Vandetanib (CAS ID 443913-73-3), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Anastassiasdis et al., Nat Biotechnol.2011 Oct. 30; 29(11): 1039-45.

In some aspects, the inhibitor is BDBM50126732(2-(2,6-Dichloro-phenylamino)-7-(3-diethylamino-propenyl)-1,6-dimethyl-1,8-dihydro-imidazo[4,5-Nisoquinolin-9-one),or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Goldberg, et al., J Med Chem, 2003;46:1337-49.

In some aspects, the inhibitor is BDBM50020476 (CHEMBL3290148), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Li, et al., J Med Chem, 2014;57:5112-28.

In some aspects, the inhibitor is BDBM4567(N-{4-[(3-bromophenyl)amino]quinazolin-6-yl}prop-2-enamide), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Tsou et al., J Med Chem, 2001;44:2719-34.

In some aspects, the inhibitor is BDBM4779(N-{4-[(3-chloro-4-fluorophenyl)amino]-743-(morpholin-4-yl)propoxy]quinazolin-6-yl}prop-2-enamide),or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Davis, et al., Nat Biotechnol, 2011;29:1046-51.

In some aspects, the inhibitor is BDBM36409 (PP242), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Davis, et al., Nat Biotechnol, 2011;29:1046-51.

In some aspects, the inhibitor is BDBM50161957 (HKI-272), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Klumpers et al., J Med Chem, 2005;48:1107-31.

In some aspects, the inhibitor is BDBM6568 (PD-173955), or apharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. See Davis, et al., Nat Biotechnol, 2011;29:1046-51.

In some embodiments, the inhibitor does not exhibit inhibitory activityagainst Itk or exhibits any such activity with an IC₅₀ value of greaterthan at or about 1000 nM or at or about 4000 or 4270 nM, or has the sameinhibitory activity towards ITK as the compound of Formula (II); doesnot inhibit TEC or exhibits any such inhibitory activity against IC₅₀only with an IC₅₀ value greater than 1000 nM or greater than 10,000 nM;does not inhibit BMX or exhibits any such inhibitory activity againstIC₅₀ only with an IC₅₀ value greater than 1000 nM or at or about orgreater than 1800 nM or greater than 10,000 nM. In some aspects, theinhibitor is not CGI-1746 (see Hendriks et al., “Targeting Bruton'styrosine kinase in B cell malignancies,” Nature, 2014, 14: 219-232;Akinleye et al., “Ibrutinib and novel BTK inhibitors in clinicaldevelopment.” Journal of Hematology & Oncology 2013, 6:59;WO2016/024230; Di Paolo et. al., Nat. Chem. Biol., 2011, 7(1): 41-50)),or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. In some embodiments, the inhibitor is notan inhibitor that exhibits activity towards ITK with an IC₅₀ value ofgreater than at or about 1000 nM or at or about 4000 or 4270 nM, or hasthe same less inhibitory activity towards ITK as the compound of Formula(II); does not inhibit TEC or exhibits any such inhibitory activityagainst IC₅₀ only with an IC₅₀ value greater than 1000 nM or greaterthan 10,000 nM; does not inhibit BMX or exhibits any such inhibitoryactivity against IC₅₀ only with an IC₅₀ value greater than 1000 nM or ator about or greater than 1800 nM or greater than 10,000 nM.

In some embodiments, the inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   Q¹ is aryl¹, heteroaryl¹, cycloalkyl, heterocyclyl,        cycloalkenyl, or heterocyclo alkenyl, any of which is optionally        substituted by one to five independent G¹ substituents;    -   R¹ is alkyl, cycloalkyl, bicycloalkyl, aryl, heteroaryl,        aralkyl, heteroaralkyl, heterocyclyl, or heterobicycloalkyl, any        of which is optionally substituted by one or more independent        G¹¹ substituents;    -   G¹ and G⁴¹ are each independently halo, oxo, —CF₃, —OCF₃, —OR²,        —NR²R³(R^(3a))_(j1), —C(O)R², —CO₂R₂, —CONR²R₃, —NO₂, —CN,        —S(O)_(j1)R², —SO₂NR²R³, NR²(C═O)R³, NR²(C═O)OR³, NR²(C═O)NR²R³,        NR²S(O)_(j1)R³, —(C═S)OR², —(C═O)SR², —NR²(C═NR³)NR^(2a)R^(3a),        —NR²(C═NR³)OR^(2a), —NR²(C═NR³)SR^(3a), —O(C═O)OR²,        —O(C═O)NR²R³, —O(C═O)SR², —S(C═O)OR², —S(C═O)NR²R³, C₀₋₁₀alkyl,        C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, C₁₋₁₀alkoxyC₂₋₁₀alkenyl,        C₁₋₁₀-alkoxyC₂₋₁₀alkynyl, C₁₋₁₀alkylthioC₁₋₁₀alkyl,        C₁₋₁₀alkylthioC₂₋₁₀alkenyl, C₁₋₁₀alkylthioC₂₋₁₀alkynyl,        cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, cycloC₃₋₈alkylC₁₋₁₀alkyl,        cycloC₃₋₈alkenylC₁₋₁₀alkyl, cycloC₃₋₈alkylC₂₋₁₀alkenyl,        cycloC₃₋₈alkenylC₂₋₁₀alkenyl, cycloC₃₋₈alkylC₂₋₁₀alkynyl,        cycloC₃₋₈alkenylC₂₋₁₀alkynyl, heterocyclyl-C₀₋₁₀alkyl,        heterocyclyl-C₂₋₁₀alkenyl, or heterocyclyl-C₂₋₁₀alkynyl, any of        which is optionally substituted with one or more independent        halo, oxo, —CF₃, —OCF₃, —OR²²², —NR²²R³³³(R^(333a))_(j1a),        C(O)R²²², —CO₂R²²², —CONR²²²R³³³, —NO₂, —CN, —S(O)_(j1a)R²²²,        SO₂NR²²²R³³³, NR²²(C═O)R³³³, NR²²²(C═O)OR³³³,        NR²²²(C═O)NR²²²R³³³, NR²²²S(O)_(j1a)R³³³, (C═S)OR²²²,        (C═O)SR²²², —NR²²²(C═NR³³³)NR^(222a)R^(333a),        —NR²²²(C═NR³³³)OR^(222a), —NR²²²(C═R³³³)SR^(333a), —O(C═O)OR²²²,        —O(C═O)NR²²²R³³³, —O(C═O)SR²²², —S(C═O)OR²²², or        —S(C═O)NR²²²R³³³ substituents; or —(X₁)_(n)—(Y¹)_(m)—R⁴; or        aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, or aryl-C₂₋₁₀alkynyl, any of        which is optionally substituted with one or more independent        halo, —CF₃, —OCF₃, —OR²²², —NR²²²R³³³(R^(333a))_(j2a),        —C(O)R²²², —CO₂R²²², CONR²²²R³³³, —NO₂, —CN, —S(O)_(j2a)R²²²,        —SO₂NR²R³³³, NR²²²(C═O)R³³³, NR²²²(C═O)OR³³³,        NR²²²(C═O)NR²²²R³³³, NR²²²S(O)_(j2a)R³³³, —(C═S)OR²²²,        —(C═O)SR²²², NR²²²(C═NR³³³)NR^(222a)R^(333a),        —NR²²²(C═NR³³³)OR^(222a), —NR²²²(C═NR³³³)SR^(333a),        —O(C═O)OR²²², —O(C═O)NR²²²R³³³, —O(C═O)SR²²², —S(C═O)OR²²², or        —S(C═O)NR²²²R³³³ substituents; or hetaryl-C₀₋₁₀alkyl,        hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, any of which is        optionally substituted with one or more independent halo, —CF₃,        —OCF₃, —OR²²², —NR²²²R³³³(R^(333a))_(j3a), —C(O)R²²², —CO₂R²²²,        CONR²²²R³³³, —NO₂, —CN, —S(O)_(j3a)R²²², —SO₂NR²²²R³³³,        NR²²²(C═O)R³³³, NR²²²(C═O)OR³³³, NR²²²(C═O)NR²²²R³³³,        NR²²²S(O)_(j3a)R³³³, —(C═S)OR²²², —(C═O)SR²²²,        —NR²²²(C═NR³³³)NR^(222a)R^(333a), —NR²²²(C═NR³³³)OR^(222a),        —NR²²²(C═NR³³³)SR^(333a), —O(C═O)OR²²², —O(C═O)NR²²²R³³³,        —O(C═O)SR²²², —S(C═O)OR²²², or —S(C═O)NR²²²R³³³ substituents;    -   G¹¹ is halo, oxo, —CF₃, —OCF₃, —OR²¹, —NR²¹R³¹(R^(3a1))_(j4),        —C(O)R²¹, —CO₂R²¹, —CONR²¹R³¹, —NO₂, —CN, —S(O)_(j4)R²¹,        —SO₂NR²¹R³¹, NR²¹(C═O)R³¹, NR²¹(C═O)OR³¹, NR²¹(C═O)NR²¹R³¹,        NR²¹S(O)_(j4)R³¹, —(C═S)OR²¹, —(C═O)SR²¹,        —NR²¹(C═NR³¹)NR^(2a1)R^(3a1), —NR²¹(C═NR³¹)OR^(2a1),        —NR²¹(C═NR³¹)SR^(3a1), —O(C═O)OR²¹, —O(C═O)NR²¹R³¹, —O(C═O)SR²¹,        —S(C═O)OR²¹, —S(C═O)NR²¹R³¹, —P(O)OR²¹OR³¹, C₀₋₁₀alkyl,        C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, C₁₋₁₀alkoxyC₁₋₁₀alkyl,        C₁₋₁₀alkoxyC₂₋₁₀alkenyl, C₁₋₁₀alkoxyC₂₋₁₀alkynyl,        C₁₋₁₀alkylthioC₁₋₁₀alkyl, C₁₋₁₀alkylthioC₂₋₁₀alkenyl,        C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl,        cycloC₃₋₈alkylC₁₋₁₀alkyl, cycloC₃₋₈alkenylC₁₋₁₀alkyl,        cycloC₃₋₈alkylC₂₋₁₀alkenyl, cycloC₃₋₈alkenylC₂₋₁₀alkenyl,        cycloC₃₋₈alkylC₂₋₁₀alkynyl, cycloC₃₋₈alkenylC₂₋₁₀alkynyl,        heterocyclyl-C₀₋₁₀alkyl, heterocyclyl-C₂₋₁₀alkenyl, or        heterocyclyl C₂₋₁₀alkynyl, any of which is optionally        substituted with one or more independent halo, oxo, —CF₃, —OCF₃,        —OR²²²¹, —NR²²²¹R³³³¹(R^(333a1))_(j4a), —C(O)R²²²¹, —CO₂R²²²¹,        —CONR²²²¹R³³³¹, —NO₂, —CN, —S(O)_(j4a)R²²²¹, —SO₂NR²²²¹R³³³¹,        NR²²²¹(C═O)R³³³¹, NR²²²¹(C═O)OR³³³¹, NR²²²¹(C═O)NR²²²¹R³³³¹,        NR²²²¹S(O)_(j4a)R³³³¹, —(C═S) R²²²¹, —(CO)SR²²²¹,        —NR²²²¹(C═NR³³³¹)NR^(222a1)R^(333a1),        —NR²²²¹(C═NR³³³¹)OR^(222a1), —NR²²²¹(C═NR³³³¹)SR^(333a1),        —O(C═O)OR²²²¹, —O(C═O)NR²²²¹R³³³¹, —O(C═O)SR²²²¹, —S(C═O)OR²²²¹,        —P(O)OR²²²¹OR³³³¹, or —S(C═O)NR²²²¹R³³³¹ substituents; or        aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, or aryl-C₂₋₁₀alkynyl, any of        which is optionally substituted with one or more independent        halo, —CF₃, —OCF₃, —OR²²²¹, —NR²²²¹R³³³¹(R^(333a1))_(j5a),        C(O)R²²²¹, —CO₂R²²²¹, —CONR²²²¹R³³³¹, —NO₂, —CN,        —S(O)_(j5a)R²²²¹, —SO₂NR²²²¹R³³³¹, NR²²²¹(C═O)R³³³¹,        NR²²²¹(C═O)OR³³³¹, NR²²²¹(C═O)NR²²²¹R³³³¹,        NR²²²¹S(O)_(j5a)R³³³¹, —(C═S)OR²²²¹, —(C═O)SR²²²¹,        —NR²²²¹(C═NR³³³¹)NR^(222a1)R^(333a1),        —NR²²²¹(C═NR³³³¹)OR^(222a1), —NR²²²¹(C═NR³³³¹)SR^(333a1),        —O(C═O)OR²²²¹, —O(C═O)NR²²²¹R³³³¹, —O(C═O)SR²²²¹, —S(C═O)OR²²²¹,        —P(O)OR²²²¹OR³³³¹, or —S(C═O)NR²²²¹R³³³¹ substituents; or        hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or        hetaryl-C₂₋₁₀alkynyl, any of which is optionally substituted        with one or more independent halo, —CF₃, —OCF₃, —OR,        —NR²²²¹R³³³¹(R^(333a1))_(j6a), —C(O)R²²²¹, —CO₂R²²²¹,        —CONR²²²¹R³³³¹, —NO₂, —CN, —S(O)_(j6a)R²²²¹,        —SO₂NR²²²¹R³³³¹NR²²²¹(C═O)R³³³¹, NR²¹(C═O)OR³³³¹,        NR²²²¹(C═O)NR²²²¹R³³³¹, NR²²¹S(O)_(j6a)R³³³¹, —(C═S)OR²²²¹,        —(CO)SR²²²¹, —NR²²²¹(C═NR³³³¹)NR^(222a1)R^(333a),        —NR²²²¹(C═NR³³³¹)OR^(222a1), —NR²²¹(C═NR³³³¹)SR^(333a1),        —O(C═O)OR²²²¹, —O(C═O)NR²²²¹R³³³¹, —O(C═O)SR²²²¹, —S(C═O)OR²²²¹,        —P(O)OR²²²¹OR³³³¹, or —S(C═O)NR²²²¹R³³³¹ substituents; or G¹¹ is        taken together with the carbon to which it is attached to form a        double bond which is substituted with R⁵ and G¹¹¹;    -   R², R^(2a), R³, R^(3a), R²²², R^(222a), R³³³, R^(333a), R²¹,        R^(2a1), R³¹, R^(3a1), R²²²¹, R^(222a1), R³³³¹, and R³³³¹ are        each independently equal to C₀₋₁₀alkyl, C₂₋₁₀alkenyl,        C₂₋₁₀alkynyl, C₁₋₁₀alkoxyC₁₋₁₀alkyl, C₁₋₁₀alkoxyC₂₋₁₀alkenyl,        C₁₋₁₀alkoxyC₂₋₁₀alkynyl, C₁₋₁₀alkylthioC₁₋₁₀alkyl,        C₁₋₁₀alkylthioC₂₋₁₀alkenyl, C₁₋₁₀alkylthioC₂₋₁₀alkynyl,        cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, cycloC₃₋₈alkylC₁₋₁₀alkyl,        cycloCs-salkenylC₁₋₁₀alkyl, cycloC₃₋₈alkylC₂₋₁₀alkenyl,        cycloC₃₋₈alkenylC₂₋₁₀alkenyl, cycloC₃₋₈alkylC₋₁₀alkynyl,        cyoloC₃₋₈alkenylC₂₋₁₀alkynyl, heterocyclyl-C₀₋₁₀alkyl,        heterocyclyl-C₂₋₁₀alkenyl, or heterocyclyl-C₂₋₁₀alkynyl, any of        which is optionally substituted by one or more G¹¹¹        substituents; or aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, or        aryl-C₂₋₁₀alkynyl, hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or        hetaryl-C₂₋₁₀alkynyl, any of which is optionally substituted by        one or more G¹¹¹ substituents; or in the case of        —NR²R³(R^(3a))_(j1) or —NR²²²R³³³(R^(333a))_(j1a) or        —NR²²²R³³³(R^(333a))_(j2a) or —NR²²²¹R³³³¹(R^(333a1))_(j3a) or        —NR²²²¹R³³³¹(R^(333a1))_(j4a) or —NR²²²¹R³³³¹(R^(333a1))_(j5a)        or —NR²²²¹R³³³¹(R^(333a1))_(j6a), R² and R³ or R²²² and R³³³ or        R²²²¹ and R³³³¹ taken together with the nitrogen atom to which        they are attached form a 3-10 membered saturated ring,        unsaturated ring, heterocyclic saturated ring, or heterocyclic        unsaturated ring, wherein said ring is optionally substituted by        one or more G¹¹¹ substituents;    -   X¹ and Y¹ are each independently —O—, —NR⁷—, —S(O)_(j7)—        —CR⁵R⁶—, —N(C(O)OR⁷)—, —N(C(O)R⁷)—, —N(SO₂R⁷)—, —CH₂O— —CH₂S—        —CH₂N(R⁷)— —CH(NR⁷)—, —CH₂N(C(O)R⁷)—, —CH₂N(C(O)OR⁷)—,        —CH₂N(SO₂R⁷)—, —CH(NHR⁷)—, —CH(NHC(O)R⁷)—, —CH(NHSO₂R⁷)—,        —CH(NHC(O)OR⁷)—, —CH(OC(O)R⁷)—, —CH(OC(O)NHR⁷)—, —CH—CH—, —C—,        —C(═NOR⁷)—, —C(O)—, —CH(OR⁷)—, —C(O)N(R⁷)—, —N(R⁷)C(O)—,        —N(R⁷)S(O)—, —N(R⁷)S(O)₂— —OC(O)N(R⁷)—, —N(R⁷)C(O)N(R⁷)—,        —NR⁷C(O)O— —S(O)N(R⁷)—, —S(O)₂N(R⁷)—, —N(C(O)R⁷)S(O)—,        —N(C(O)R⁷)S(O)₂—, —N(R⁷)S(O)N(R⁷)—, —N(R⁷)S(O)₂N; (R⁷)—,        —C(O)N(R⁷)C(O)—, —S(O)N(R⁷)C(O)—, —S(O)₂N(R⁷)C(O)—,        —OS(O)N(R⁷)—, —OS(O)₂N(R⁷)—, —N(R⁷)S(O)O—, —N(R⁷)S(O)₂O—,        —N(R⁷)S(O)C(O)— —N(R⁷)S(O)₂C(O)—, —SON(C(O)R⁷)—, —SO₂N(C(O)R⁷)—,        —N(R⁷)SON(R⁷)—, —N(R⁷)SO₂N(R⁷)—, —C(O)O— —N(R⁷)P(OR⁸)O—,        —N(R⁷)P(OR⁸)—, —N(R⁷)P(O)(OR⁸)O— —N(R⁷)P(O)(OR⁸)—,        —N(C(O)R⁷)P(OR⁸)O— —N(C(O)R⁷)P(OR⁸)—, —N(C(O)R⁷)P(O)(OR⁸)O—,        —N(C(O)R⁷)P(OR⁸)—, —CH(R⁷)S(O)—, —CH(R⁷)S(O)₂—        —CH(R⁷)N(C(O)OR⁷)— —CH(R⁷)N(C(O)R⁷)—, —CH(R⁷)N(SO₂R⁷)—,        —CH(R⁷)O— —CH(R⁷)S— —CH(R⁷)N(R⁷)—, —CH(R⁷)N(C(O)R⁷)—,        —CH(R⁷)N(C(O)OR⁷)—, —CH(R⁷)N(SO₂R⁷)—, —CH(R⁷)C(═NOR⁷)—,        —CH(R⁷)C(O)—, —CH(R⁷)CH(OR⁷)—, —CH(R⁷)C(O)N(R⁷)—,        —CH(R⁷)N(R⁷)C(O)—, —CH(R⁷)N(R⁷)S(O)—, —CH(R⁷)N(R⁷)S(O)₂—,        —CH(R⁷)OC(O)N(R⁷)—, —CH(R⁷)N(R⁷)C(O)N(R⁷)—, —CH(R⁷)NR⁷C(O)O—,        —CH(R⁷)S(O)N(R⁷)—, —CH(R⁷)S(O)₂N(R⁷)—, —CH(R⁷)N(C(O)R⁷)S(O)—,        —CH(R⁷)N(C(O)R⁷)S(O)—, —CH(R⁷)N(R⁷)S(O)N(R⁷)—,        —CH(R⁷)N(R⁷)S(O)₂N(R⁷)—, —CH(R⁷)C(O)N(R⁷)C(O)—,        —CH(R⁷)S(O)N(R⁷)C(O)—, —CH(R⁷)S(O)₂N(R⁷)C(O)—        —CH(R⁷)OS(O)N(R⁷)—, —CH(R⁷)OS(O)₂N(R⁷)—, —CH(R⁷)N(R⁷)S(O)O—,        —CH(R⁷)N(R⁷)S(O)₂O—, —CH(R⁷)N(R⁷)S(O)C(O)—,        —CH(R⁷)N(R⁷)S(O)₂C(O)—, —CH(R⁷)SON(C(O)R⁷)—,        —CH(R⁷)SO₂N(C(O)R⁷)—, —CH(R⁷)N(R⁷)SON(R⁷)—,        —CH(R⁷)N(R⁷)SO₂N(R⁷)—, —CH(R⁷)C(O)O—, —CH(R⁷)N(R⁷)P(OR⁸)O—        —CH(R⁷)N(R⁷)P(OR⁸)—, —CH(R⁷)N(R⁷)P(O)(OR⁸)O—,        —CH(R⁷)N(R⁷)P(O)(OR⁸)—, —CH(R⁷)N(C(O)R⁷)P(OR⁸)O—,        —CH(R⁷)N(C(O)R⁷)P(OR⁸)— —CH(R⁷)N(C(O)R⁷)P(O)(OR⁸)O—, or        —CH(R⁷)N(C(O)R⁷)P(OR⁸)—; or    -   X¹ and Y¹ are each independently represented by one of the        following structural formulas:

-   -   R¹⁰, taken together with the phosphinamide or phosphonamide, is        a 5-, 6-, or 7-membered aryl, heteroaryl or heterocyclyl ring        system;    -   R⁵, R⁶, and G¹¹¹ are each independently a C₀₋₁₀alkyl,        C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, C₁₋₁₀alkoxyC₁₋₁₀alkyl,        C₁₋₁₀alkoxyC₂₋₁₀alkenyl, C₁₋₁₀alkoxyC₂₋₁₀alkynyl,        C₁₋₁₀alkylthioC₁₋₁₀alkyl, C₁₋₁₀alkylthioC₂₋₁₀alkenyl,        C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl,        cycloC₃₋₈alkylC₁₋₁₀alkyl, cycloC₃₋₈alkenylC₁₋₁₀alkyl,        cycloC₃₋₈alkylC₂₋₁₀alkenyl, cyclOC₃₋₈alkenylC₂₋₁₀alkenyl,        cycloC₃₋₈alkylC₂₋₁₀alkynyl, cycloC₃₋₈alkenylC₂₋₁₀alkynyl,        heterocyclyl-C₀₋₁₀alkyl, heterocyclyl-C₂₋₁₀alkenyl, or        heterocyclyl-C₂₋₁₀alkynyl, any of which is optionally        substituted with one or more independent halo, —CF₃, —OCF₃,        —OR⁷⁷, —NR⁷⁷R⁸⁷, —C(O)R⁷⁷, —CO₂R⁷⁷, —CONR⁷⁷R⁸⁷, —NO₂, —CN,        —S(O)_(j5a)R⁷⁷, —SO₂NR⁷⁷R⁸⁷, NR⁷⁷(C═O)R⁸⁷, NR⁷⁷(C═O)OR⁸⁷,        NR⁷⁷(C═O)NR⁷⁸R⁸⁷, NR⁷⁷S(O)_(j5a)R⁸⁷, —(C═S)OR⁷⁷, —(C═O)SR⁷⁷,        —NR⁷⁷(C═NR⁸⁷)R⁷⁸R⁸⁸, —NR⁷⁷(C═NR⁸⁷)OR⁷⁸, —NR⁷⁷(C═NR⁸⁷)SR⁷⁸,        —O(C═O)OR⁷⁷, —O(C═O)NR⁷⁷R⁸⁷, —O(C═O)SR⁷⁷, —S(C═O)OR⁷⁷,        —P(O)OR⁷⁷OR⁸⁷, or —S(C═O)NR⁷⁷R⁸⁷ substituents; or        aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, or aryl-C₂₋₁₀alkynyl, any of        which is optionally substituted with one or more independent        halo, —CF₃, —OCF₃, —OR⁷⁷, —NR⁷⁷R⁸⁷, —C(O)R⁷⁷, —CO₂R⁷⁷,        —CONR⁷⁷R⁸⁷, —NO₂, —CN, —S(O)_(j5a)R⁷⁷, —SO₂NR⁷⁷R⁸⁷,        NR⁷⁷(C═O)R⁸⁷, NR⁷⁷(C═O)OR⁸⁷, NR⁷⁷(C═O)NR⁷⁸R⁸⁷,        NR⁷⁷S(O)_(j5a)R⁸⁷, —(C═S)OR⁷⁷, —(C═O)SR⁷⁷, —NR⁷⁷(C═NR⁸⁷)NR⁷⁸R⁸⁸,        —NR⁷⁷(C═NR⁸⁷)OR⁷⁸, —NR⁷⁷(C═NR⁸⁷)SR⁷⁸, —O(C═O)OR⁷⁷,        —O(C═O)NR⁷⁷R⁸⁷, —O(C═O)SR⁷⁷, —S(C═O)OR⁷⁷, —P(O)OR⁷⁷OR⁸⁷, or        —S(C═O)NR⁷⁷R⁸⁷ substituents; or hetaryl-C₀₋₁₀alkyl,        hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, any of which is        11 optionally substituted with one or more independent halo,        —CF, —OCF₃, —OR, —NR⁷⁷R⁸⁷, —C(O)R⁷⁷, —CO₂R⁷⁷, —CONR⁷⁷R⁸⁷, —NO₂,        —CN, —S(O)_(j5a)R⁷⁷, —SO₂NR⁷⁷R⁸⁷, NR⁷⁷(C═O)R⁸⁷, NR⁷⁷(C═O)OR⁸⁷,        NR⁷⁷(C═O)NR⁷⁸R⁸⁷, NR⁷⁷S(O)_(j5a)R⁸⁷, —(C═S)OR⁷⁷, —(C═O)SR⁷⁷,        —NR⁷⁷(C═NR⁸⁷)NR⁷⁸R⁸⁸, —NR⁷⁷(C═NR⁸⁷)OR⁷⁸, —NR⁷⁷(C═NR⁸⁷)SR⁷⁸,        —O(C═O)OR⁷⁷, —O(C═O)NR⁷⁷R⁸⁷, —O(C═O)SR⁷⁷, —S(C═O)OR⁷⁷,        —P(O)OR⁷⁷OR⁸⁷, or —S(C═O)NR⁷⁷R⁸⁷ substituents; or R⁵ with R⁶        taken together with the respective carbon atom to which they are        attached, form a 3-10 membered saturated or unsaturated ring,        wherein said ring is optionally substituted with R⁶⁹; or R⁵ with        R⁶ taken together with the respective carbon atom to which they        are attached, form a 3-10 membered saturated or unsaturated        heterocyclic ring, wherein said ring is optionally substituted        with R⁶⁹;    -   R⁷ and R⁸ are each independently H, acyl, alkyl, alkenyl, aryl,        heteroaryl, heterocyclyl or cycloalkyl, any of which is        optionally substituted by one or more G¹¹ substituents;    -   R⁴ is H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,        heterocyclyl, cycloalkenyl, or heterocycloalkenyl, any of which        is optionally substituted by one or more G⁴¹ substituents;    -   R⁶⁹ is halo, —OR⁷⁸, —SH, —NR⁷⁸R⁸⁸, —CO₂R⁷⁸, —CONR⁷⁸R⁸⁸, —NO₂,        —CN, —S(O)_(j8)R⁷⁸, —SO₂NR⁷⁸R⁸⁸, C₀₋₁₀alkyl, C₂₋₁₀alkenyl,        C₂₋₁₀alkynyl, C₁₋₁₀alkoxyC₁₋₁₀alkyl, C₁₋₁₀alkoxyC₂₋₁₀alkenyl,        C₁₋₁₀alkoxyC₂₋₁₀alkynyl, C₁₋₁₀alkylthioC₁₋₁₀alkyl,        C₁₋₁₀alkylthioC₂₋₁₀alkenyl, C₁₋₁₀alkylthioC₂₋₁₀alkynyl,        cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl, cycloC₃₋₈alkylC₁₋₁₀alkyl,        cycloC₃₋₈alkenylC₁₋₁₀alkyl, cyclOC₃₋₈alkylC₂₋₁₀alkenyl,        cycloC₃₋₈alkenylC₂₋₁₀alkenyl, cyclOC₃₋₈alkylC₂₋₁₀alkynyl,        cycloC₃₋₈alkenylC₂₋₁₀alkynyl, heterocyclyl-C₀₋₁₀alkyl,        heterocyclyl-C₂₋₁₀alkenyl, or heterocyclyl-C₂₋₁₀alkynyl, any of        which is optionally substituted with one or more independent        halo, cyano, nitro, —OR⁷⁷⁸, —SO₂NR⁷⁷⁸R⁸⁸⁸, or —NR⁷⁷⁸R⁸⁸⁸        substituents; or aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, or        aryl-C₂₋₁₀alkynyl, any of which is optionally substituted with        one or more independent halo, cyano, nitro, —OR⁷⁷⁸, C₁₋₁₀alkyl,        C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, haloC₁₋₁₀alkyl, haloC₂₋₁₀alkenyl,        haloC₂₋₁₀alkynyl, —COOH, C₁₋₄alkoxycarbonyl, —CONR⁷⁸R⁸⁸⁸,        —SO₂NR⁷⁷⁸R⁸⁸⁸, or —NR⁷⁷⁸R⁸⁸⁸ substituents; or        hetaryl-C₀₋₁₀alkyl, hetaryl-C₂₋₁₀alkenyl, or        hetaryl-C₂₋₁₀alkynyl, any of which is optionally substituted        with one or more independent halo, cyano, nitro, —OR⁷⁷⁸,        C₁₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, haloC₁₋₁₀alkyl,        haloC₂₋₁₀alkenyl, haloC₂₋₁₀alkynyl, —COOH, C₁₋₄alkoxycarbonyl,        —CONR⁷⁷⁸R⁸⁸⁸, —SO₂NR⁷⁷⁸R⁸⁸⁸, or —NR⁷⁷⁸R⁸⁸⁸ substituents; or        mono(C₁₋₆alkyl)aminoC₁₋₆alkyl, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,        mono(aryl)aminoC₁₋₆alkyl, di(aryl)aminoC₁₋₆alkyl, or        —N(C₁₋₆alkyl)-C₁₋₆alkyl-aryl, any of which is optionally        substituted with one or more independent halo, cyano, nitro,        —OR⁷⁷⁸, C₁₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, haloC₁₋₁₀alkyl,        haloC₂₋₁₀alkenyl, haloC₂₋₁₀alkynyl, —COOH, C₁₋₄alkoxycarbonyl,        —CONR⁷⁷⁸R⁸⁸⁸, —SO₂NR⁷⁷⁸R⁸⁸⁸, or —NR⁷⁷⁸R⁸⁸⁸ substituents; or in        the case of —NR⁷⁸R⁸⁸, R⁷⁸ and R⁸⁸ taken together with the        nitrogen atom to which they are attached form a 3-10 membered        saturated ring, unsaturated ring, heterocyclic saturated ring,        or heterocyclic unsaturated ring, wherein said ring is        optionally substituted with one or more independent halo, cyano,        hydroxy, nitro, C₁₋₁₀alkoxy, —SO₂NR⁷⁷⁸R⁸⁸⁸, or —NR⁷⁷⁸R⁸⁸⁸        substituents;    -   R⁷⁷, R⁷⁸, R⁸⁷, R⁸⁸, R⁷⁷⁸, and R⁸⁸⁸ are each independently        C₀₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, C₁₋₁₀alkoxyC₁₋₁₀alkyl,        C₁₋₁₀alkoxyC₂₋₁₀alkenyl, C₁₋₁₀alkoxyC₂₋₁₀alkynyl,        C₁₋₁₀alkylthioC₁₋₁₀alkyl, C₁₋₁₀alkylthioC₂₋₁₀alkenyl,        C₁₋₁₀alkylthioC₂₋₁₀alkynyl, cycloC₃₋₈alkyl, cycloC₃₋₈alkenyl,        cycloC₃₋₈alkylC₁₋₁₀alkyl, cycloC₃₋₈alkenylC₁₋₁₀alkyl,        cycloC₃₋₈alkylC₂₋₁₀alkenyl, cycloC₃₋₈alkenylC₂₋₁₀alkenyl,        cycloC₃₋₈alkylC₂₋₁₀alkynyl, cycloC₃₋₈alkenylC₂₋₁₀alkynyl,        heterocyclyl-C₀₋₁₀alkyl, heterocyclyl-C₂₋₁₀alkenyl,        heterocyclyl-C₂₋₁₀alkynyl, C₁₋₁₀alkylcarbonyl,        C₂₋₁₀alkenylcarbonyl, C₂₋₁₀alkynylcarbonyl, C₁₋₁₀alkoxycarbonyl,        C₁₋₁₀alkoxycarbonylC₁₋₁₀alkyl, monoC₁₋₆alkylaminocarbonyl,        di-C₁₋₆alkylaminocarbonyl, mono(aryl)aminocarbonyl,        di(aryl)aminocarbonyl, or C₁₋₁₀alkyl(aryl)aminocarbonyl, any of        which is optionally substituted with one or more independent        halo, cyano, hydroxy, nitro, C₁₋₁₀alkoxy,        —SO₂N(C₀₋₄alkyl)(C₀₋₄alkyl), or —N(C₀₋₄alkyl)(C₀₋₄alkyl)        substituents; or aryl-C₀₋₁₀alkyl, aryl-C₂₋₁₀alkenyl, or        aryl-C₂₋₁₀alkynyl, any of which is optionally substituted with        one or more independent halo, cyano, nitro, —O(C₀₋₄alkyl),        C₁₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl, haloC₁₋₁₀alkyl,        haloC₂₋₁₀alkenyl, haloC₂₋₁₀alkynyl, —COOH, C₁₋₄alkoxycarbonyl,        —CON(C₀₋₄alkyl)(C₀₋₁₀alkyl), —SO₂N(C₀₋₄alkyl)(C₀₋₄alkyl), or        —N(C₀₋₄alkyl)(C₀₋₄alkyl) substituents; or hetaryl-C₀₋₁₀alkyl,        hetaryl-C₂₋₁₀alkenyl, or hetaryl-C₂₋₁₀alkynyl, any of which is        optionally substituted with one or more independent halo, cyano,        nitro, —O(C₀₋₄alkyl), C₁₋₁₀alkyl, C₂₋₁₀alkenyl, C₂₋₁₀alkynyl,        haloC₁₋₁₀alkyl, haloC₂₋₁₀alkenyl, haloC₂₋₁₀alkynyl, —COOH,        C₁₋₄alkoxycarbonyl, —CON(C₀₋₄alkyl)(C₀₋₄alkyl),        —SO₂N(C₀₋₄alkyl)(C₀₋₄alkyl), or —N(C₀₋₄alkyl)(C₀₋₄alkyl)        substituents; or mono(C₁₋₆alkyl)aminoC₁₋₆alkyl,        di(C₁₋₆alkyl)aminoC₁₋₆alkyl, mono(aryl)aminoC₁₋₆alkyl,        di(aryl)aminoC₁₋₆alkyl, or —N(C₁₋₆alkyl)-C₁₋₆alkyl-aryl, any of        which is optionally substituted with one or more independent        halo, cyano, nitro, —O(C₀₋₄alkyl), C₁₋₁₀alkyl, C₂₋₁₀alkenyl,        C₂₋₁₀alkynyl, haloC₁₋₁₀alkyl, haloC₂₋₁₀alkenyl,        haloC₂₋₁₀alkynyl, —COOH, C₁₋₄alkoxycarbonyl,        —CON(C₀₋₄alkyl)(C₀₋₄alkyl), —SO₂N(C₀₋₄alkyl)(C₀₋₄alkyl), or        —N(C₀₋₄alkyl)(C₀₋₄alkyl) substituents; and n, m, j1, j1a, j2a,        j3a, j4, j4a, j5a, j6a, j7, and j8 are each independently equal        to 0, 1, or 2.

In some embodiments, the inhibitor is4-{8-Amino-3-[(2S)-1-(2-butynoyl)-2-pyrrolidinyl]imidazo[1,5-a]pyrazin-1-yl}-N-(2-pyridinyl)benzamide,or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof. In some embodiments, the inhibitor has theCAS Number 1420477-60-6.

In some embodiments, the inhibitor is a compound of Formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof.

In some embodiments, the inhibitor is an inhibitor as described in U.S.Pat. No. 7,459,554 and PCT Application No. WO2005/037836.

1. Compositions and Formulations

In some embodiments of the methods, compositions, combinations, kits anduses provided herein, the combination therapy can be administered in oneor more compositions, e.g., a pharmaceutical composition containing aninhibitor of a TEC family kinase, e.g. a Btk inhibitor, and/or the celltherapy, e.g., T cell therapy.

In some embodiments, the composition, e.g., a pharmaceutical compositioncontaining an inhibitor of a protein tyrosine kinase other than aninhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, can include carriers such as a diluent,adjuvant, excipient, or vehicle with which the inhibitor, and/or thecells are administered. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions will contain a therapeutically effective amount of thetyrosine kinase inhibitor, e.g. Btk inhibitor, generally in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, and sesame oil. Saline solutions and aqueousdextrose and glycerol solutions also can be employed as liquid carriers,particularly for injectable solutions. The pharmaceutical compositionscan contain any one or more of a diluents(s), adjuvant(s),antiadherent(s), binder(s), coating(s), filler(s), flavor(s), color(s),lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s),emulsifying agent(s), pharmaceutical excipient(s), pH bufferingagent(s), or sweetener(s) and a combination thereof. In someembodiments, the pharmaceutical composition can be liquid, solid, alyophilized powder, in gel form, and/or combination thereof. In someaspects, the choice of carrier is determined in part by the particularinhibitor and/or by the method of administration.

Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG), stabilizers and/or preservatives. The compositions containing thetyrosine kinase inhibitor, e.g. Btk inhibitor can also be lyophilized.

In some embodiments, the pharmaceutical compositions can be formulatedfor administration by any route known to those of skill in the artincluding intramuscular, intravenous, intradermal, intralesional,intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal,oral, vaginal, rectal, topical, local, otic, inhalational, buccal (e.g.,sublingual), and transdermal administration or any route. In someembodiments, other modes of administration also are contemplated. Insome embodiments, the administration is by bolus infusion, by injection,e.g., intravenous or subcutaneous injections, intraocular injection,periocular injection, subretinal injection, intravitreal injection,trans-septal injection, subscleral injection, intrachoroidal injection,intracameral injection, subconjectval injection, subconjuntivalinjection, sub-Tenon's injection, retrobulbar injection, peribulbarinjection, or posterior juxtascleral delivery. In some embodiments,administration is by parenteral, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In some embodiments, agiven dose is administered by a single bolus administration. In someembodiments, it is administered by multiple bolus administrations, forexample, over a period of no more than 3 days, or by continuous infusionadministration.

In some embodiments, the administration can be local, topical orsystemic depending upon the locus of treatment. In some embodimentslocal administration to an area in need of treatment can be achieved by,for example, but not limited to, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant. In some embodiments, compositions also can beadministered with other biologically active agents, either sequentially,intermittently or in the same composition. In some embodiments,administration also can include controlled release systems includingcontrolled release formulations and device controlled release, such asby means of a pump. In some embodiments, the administration is oral.

In some embodiments, pharmaceutically and therapeutically activecompounds and derivatives thereof are typically formulated andadministered in unit dosage forms or multiple dosage forms. Each unitdose contains a predetermined quantity of therapeutically activecompound sufficient to produce the desired therapeutic effect, inassociation with the required pharmaceutical carrier, vehicle ordiluent. In some embodiments, unit dosage forms, include, but are notlimited to, tablets, capsules, pills, powders, granules, sterileparenteral solutions or suspensions, and oral solutions or suspensions,and oil water emulsions containing suitable quantities of the compoundsor pharmaceutically acceptable derivatives thereof. Unit dose forms canbe contained ampoules and syringes or individually packaged tablets orcapsules. Unit dose forms can be administered in fractions or multiplesthereof. In some embodiments, a multiple dose form is a plurality ofidentical unit dosage forms packaged in a single container to beadministered in segregated unit dose form. Examples of multiple doseforms include vials, bottles of tablets or capsules or bottles of pintsor gallons.

2. Inhibitor Dosage Schedule

In some embodiments, the method involves administering to the subject atherapeutically effective amount of an inhibitor of a protein tyrosinekinase other than an inhibitor of ITK and/or an inhibitor of one or moreof BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4, and the cell therapy, suchas a T cell therapy (e.g. CAR-expressing T cells) or a T cell-engagingtherapy. In some embodiments, the inhibitor, is administered prior to,subsequently to, during, during the course of, simultaneously, nearsimultaneously, sequentially and/or intermittently with theadministration of the cell therapy, such as a T cell therapy (e.g.CAR-expressing T cells) or a T cell-engaging therapy. In someembodiments, the method involves administering the inhibitor, prior toadministration of the T cell therapy. In other embodiments, the methodinvolves administering the inhibitor, after administration of the T celltherapy. In some embodiments, the inhibitor, is not further administeredafter initiation of the T cell therapy. In some embodiments, the dosageschedule comprises administering the inhibitor o, prior to and afterinitiation of the T cell therapy. In some embodiments, the dosageschedule comprises administering the inhibitor, simultaneously with theadministration of the T cell therapy.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, is administered multiple times inmultiple doses. In some embodiments, the inhibitor, is administeredonce. In some embodiments, the inhibitor, is administered six timesdaily, five times daily, four times daily, three times daily, twicedaily, once daily, every other day, every three days, twice weekly, onceweekly or only one time prior to or subsequently to initiation ofadministration of the cell therapy (e.g. T cell therapy, such as CAR-Tcell therapy). In some embodiments, the inhibitor is administered inmultiple doses in regular intervals prior to, during, during the courseof, and/or after the period of administration of the cell therapy (e.g.T cell therapy, such as CAR-T cell therapy). In some embodiments, theinhibitor, is administered in one or more doses in regular intervalsprior to the administration of the cell therapy (e.g. T cell therapy,such as CAR-T cell therapy). In some embodiments, the inhibitor isadministered in one or more doses in regular intervals after theadministration of the cell therapy (e.g. T cell therapy, such as CAR-Tcell therapy). In some embodiments, one or more of the doses of theinhibitor can occur simultaneously with the administration of a dose ofthe cell therapy (e.g. T cell therapy, such as CAR-T cell therapy).

In some embodiments, the dose, frequency, duration, timing and/or orderof administration of the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, is determined, based on particularthresholds or criteria of results of the screening step and/orassessment of treatment outcomes described herein, e.g., those describedin Section IV herein.

In some embodiments, the method involves administering the cell therapyto a subject that has been previously administered a therapeuticallyeffective amount of the inhibitor. In some embodiments, the inhibitor isadministered to a subject before administering a dose of cellsexpressing a recombinant receptor to the subject. In some embodiments,the treatment with the inhibitor occurs at the same time as theadministration of the dose of cells. In some embodiments, the inhibitoris administered after the administration of the dose of cells. In someembodiments, the inhibitor is administered at a sufficient time prior tocell therapy so that the therapeutic effect of the combination therapyis increased.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, is administered prior to and/orconcurrently with the administration of the cell therapy (e.g. T celltherapy, such as CAR-T cell therapy). In some embodiments, the inhibitoris administered from or from about 0 to 90 days, such as 0 to 30 days, 0to 15 days, 0 to 6 days, 0 to 96 hours, 0 to 24 hours, 0 to 12 hours, 0to 6 hours, or 0 to 2 hours, 2 hours to 30 days, 2 hours to 15 days, 2hours to 6 days, 2 hours to 96 hours, 2 hours to 24 hours, 2 hours to 12hours, 2 hours to 6 hours, 6 hours to 90 days, 6 hours to 30 days, 6hours to 15 days, 6 hours to 6 days, 6 hours to 96 hours, 6 hours to 24hours, 6 hours to 12 hours, 12 hours to 90 days, 12 hours to 30 days, 12hours to 15 days, 12 hours to 6 days, 12 hours to 96 hours, 12 hours to24 hours, 24 hours to 90 days, 24 hours to 30 days, 24 hours to 15 days,24 hours to 6 days, 24 hours to 96 hours, 96 hours to 90 days, 96 hoursto 30 days, 96 hours to 15 days, 96 hours to 6 days, 6 days to 90 days,6 days to 30 days, 6 days to 15 days, 15 days to 90 days, 15 days to 30days or 30 days to 90 days prior to initiation of the cell therapy (e.g.T cell therapy, such as CAR-T cell therapy). In some aspects, theinhibitor is administered no more than about 96 hours, 72 hours, 48hours, 24 hours, 12 hours, 6 hours, 2 hours or 1 hour prior toinitiation of the cell therapy (e.g. T cell therapy, such as CAR-T celltherapy).

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, is administered at least or about atleast 1 hours, at least or about at least 2 hours, at least or about atleast 6 hours, at least or about at least 12 hours, at least or about atleast 1 day, at least or about at least 2 days, at least or about atleast 3 days, at least or about at least 4 days, at least or about atleast 5 days, at least or about at least 6 days, at least or about atleast 7 days, at least or at least about 12 days, at least or about atleast 14 days, at least or at least about 15 days, at least or about atleast 21 days, at least or at least about 24 days, at least or about atleast 28 days, at least or about at least 30 days, at least or about atleast 35 days or at least or about at least 42 days, at least or aboutat least 60 days, or at least or about at least 90 days prior toinitiation of the administration of the cell therapy (e.g. T celltherapy, such as a CAR-T cell therapy). In some embodiments, theinhibitor of the TEC family kinase, e.g., a BTK inhibitor, isadministered up to 2 days, up to 3 days, up to 4 days, up to 5 days, upto 6 days, up to 7 days, up to 8 days, up to 12 days, up to 14 days, upto 15 days, up to 21 days, up to 24 days, up to 28 days, up to 30 days,up to 35 days, up to 42 days, up to 60 days or up to 90 days prior toinitiation of administration of the cell therapy (e.g. T cell therapy,such as CAR-T cell therapy).

In some of any such embodiments in which the inhibitor of a proteintyrosine kinase other than an inhibitor of ITK and/or an inhibitor ofone or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4 is given prior tothe cell therapy (e.g. T cell therapy, such as CAR-T cell therapy), theadministration of the inhibitor continues at regular intervals until theinitiation of the cell therapy and/or for a time after the initiation ofthe cell therapy.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 is administered, or is furtheradministered, after administration of the cell therapy (e.g. T celltherapy, such as CAR-T cell therapy). In some embodiments, the inhibitoris administered within or within about 1 hours, 2 hours, 6 hours, 12hours, 24 hours, 48 hours, 96 hours, 4 days, 5 days, 6 days or 7 days,14 days, 15 days, 21 days, 24 days, 28 days, 30 days, 36 days, 42 days,60 days, 72 days or 90 days after initiation of administration of thecell therapy (e.g. T cell therapy). In some embodiments, the providedmethods involve continued administration, such as at regular intervals,of the inhibitor after initiation of administration of the cell therapy.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4r, is administered, such as is administereddaily, for up to or up to about 1 day, up to or up to about 2 days, upto or up to about 3 days, up to or up to about 4 days, up to or up toabout 5 days, up to or up to about 6 days, up to or up to about 7 days,up to or up to about 12 days, up to or up to about 14 days, up to or upto about 21 days, up to or up to about 24 days, up to or up to about 28days, up to or up to about 30 days, up to or up to about 35 days, up toor up to about 42 days, up to or up to about 60 days or up to or up toabout 90 days, up to or up to about 120 days, up to or up to about 180days, up to or up to about 240 days, up to or up about 360 days, or upto or up to about 720 days or more after the administration of the celltherapy (e.g. T cell therapy, such as CAR-T cell therapy).

In some of any such above embodiments, the inhibitor of a proteintyrosine kinase other than an inhibitor of ITK and/or an inhibitor ofone or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4, is administeredprior to and after initiation of administration of the cell therapy(e.g. T cell therapy, such as CAR-T cell therapy).

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 is administered several times a day, twicea day, daily, every other day, three times a week, twice a week, or oncea week after initiation of the cell therapy. In some embodiments, theinhibitor is administered daily. In some embodiments the inhibitor isadministered twice a day. In some embodiments, the inhibitor isadministered three times a day. In other embodiments, the inhibitor isadministered every other day.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 is administered daily for a cycle of 7,14, 21, 28, 35, or 42 days. In some embodiments, the inhibitor isadministered twice a day for a cycle of 7, 14, 21, 28, 35, or 42 days.In some embodiments, the inhibitor is administered three times a day fora cycle of 7, 14, 21, 28, 35, or 42 days. In some embodiments, theinhibitor is administered every other day for a cycle of 7, 14, 21, 28,35, or 42 days. In some embodiments, the inhibitor is administered for1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, or 24 cycles.

In some embodiments of the methods provided herein, the inhibitor of aprotein tyrosine kinase other than an inhibitor of ITK and/or aninhibitor of one or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4, andthe cell therapy (e.g. T cell therapy, such as CAR-T cell therapy) areadministered simultaneously or near simultaneously.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, is independently administered in a dosageamount of from or from about 0.2 mg per kg body weight of the subject(mg/kg) to 200 mg/kg, 0.2 mg/kg to 100 mg/kg, 0.2 mg/kg to 50 mg/kg, 0.2mg/kg to 10 mg/kg, 0.2 mg/kg to 1.0 mg/kg, 1.0 mg/kg to 200 mg/kg, 1.0mg/kg to 100 mg/kg, 1.0 mg/kg to 50 mg/kg, 1.0 mg/kg to 10 mg/kg, 10mg/kg to 200 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, 50mg/kg to 200 mg/kg, 50 mg/kg to 100 mg/kg or 100 mg/kg to 200 mg/kg. Insome embodiments, the inhibitor is administered at a dose of about 0.2mg per kg body weight of the subject (mg/kg) to 50 mg/kg, 0.2 mg/kg to25 mg/kg, 0.2 mg/kg to 10 mg/kg, 0.2 mg/kg to 5 mg/kg, 0.2 mg/kg to 1.0mg/kg, 1.0 mg/kg to 50 mg/kg, 1.0 mg/kg to 25 mg/kg, 1.0 mg/kg to 10mg/kg, 1.0 mg/kg to 5 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 25 mg/kg, 5mg/kg to 10 mg/kg, or 10 mg/kg to 25 mg/kg.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, is independently administered in a dosageamount of from or from about 25 mg to 2000 mg, 25 mg to 1000 mg, 25 mgto 500 mg, 25 mg to 200 mg, 25 mg to 100 mg, 25 mg to 50 mg, 50 mg to2000 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, 50 mg to100 mg, 100 mg to 2000 mg, 100 mg to 1000 mg, 100 mg to 500 mg, 100 mgto 200 mg, 200 mg to 2000 mg, 200 mg to 1000 mg, 200 mg to 500 mg, 500mg to 2000 mg, 500 mg to 1000 mg or 1000 mg to 2000 mg, each inclusive.

In some embodiments, the inhibitor is the compound of Formula (II),which is administered, in a dosage amount of from or from about 50 mg to420 mg, 50 mg to 400 mg, 50 mg to 380 mg, 50 mg to 360 mg, 50 mg to 340mg, 50 mg to 320 mg, 50 mg to 300 mg, 50 mg to 280 mg, 100 mg to 400 mg,100 mg to 380 mg, 100 mg to 360 mg, 100 mg to 340 mg, 100 mg to 320 mg,100 mg to 300 mg, 100 mg to 280 mg, 100 mg to 200 mg, 140 mg to 400 mg,140 mg to 380 mg, 140 mg to 360 mg, 140 mg to 340 mg, 140 mg to 320 mg,140 mg to 300 mg, 140 mg to 280 mg, 140 mg to 200 mg, 180 mg to 400 mg,180 mg to 380 mg, 180 mg to 360 mg, 180 mg to 340 mg, 180 mg to 320 mg,180 mg to 300 mg, 180 mg to 280 mg, 200 mg to 400 mg, 200 mg to 380 mg,200 mg to 360 mg, 200 mg to 340 mg, 200 mg to 320 mg, 200 mg to 300 mg,200 mg to 280 mg, 220 mg to 400 mg, 220 mg to 380 mg, 220 mg to 360 mg,220 mg to 340 mg, 220 mg to 320 mg, 220 mg to 300 mg, 220 mg to 280 mg,240 mg to 400 mg, 240 mg to 380 mg, 240 mg to 360 mg, 240 mg to 340 mg,240 mg to 320 mg, 240 mg to 300 mg, 240 mg to 280 mg, 280 mg to 420 mg,or 300 mg to 400 mg, each inclusive.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4, is administered at a total daily dosageamount of at least or at least about 50 mg/day, 100 mg/day, 150 mg/day,175 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 350 mg/day, 400 mg/day,420 mg/day, 440 mg/day, 460 mg/day, 480 mg/day, 500 mg/day, 520 mg/day,540 mg/day, 560 mg/day, 580 mg/day or 600 mg/day. In some embodiments,the inhibitor is administered in an amount less than 420 mg/day. In someembodiments, the inhibitor is administered once daily. In someembodiments, the inhibitor is administered twice daily.

In any of the aforementioned embodiments, the inhibitor, e.g. thecompound of Formula (II), may be administered orally.

In some embodiments, dosages, such as daily dosages, are administered inone or more divided doses, such as 2, 3, or 4 doses, or in a singleformulation. The inhibitor can be administered alone, in the presence ofa pharmaceutically acceptable carrier, or in the presence of othertherapeutic agents.

One skilled in the art will recognize that higher or lower dosages ofthe inhibitor could be used, for example depending on the particularagent and the route of administration. In some embodiments, theinhibitor may be administered alone or in the form of a pharmaceuticalcomposition wherein the compound is in admixture or mixture with one ormore pharmaceutically acceptable carriers, excipients, or diluents. Insome embodiments, the inhibitor may be administered either systemicallyor locally to the organ or tissue to be treated. Exemplary routes ofadministration include, but are not limited to, topical, injection (suchas subcutaneous, intramuscular, intradermal, intraperitoneal,intratumoral, and intravenous), oral, sublingual, rectal, transdermal,intranasal, vaginal and inhalation routes. In some embodiments, theroute of administration is oral, parenteral, rectal, nasal, topical, orocular routes, or by inhalation. In some embodiments, the inhibitor isadministered orally. In some embodiments, the inhibitor is administeredorally in solid dosage forms, such as capsules, tablets and powders, orin liquid dosage forms, such as elixirs, syrups and suspensions.

Once improvement of the patient's disease has occurred, the dose may beadjusted for preventative or maintenance treatment. For example, thedosage or the frequency of administration, or both, may be reduced as afunction of the symptoms, to a level at which the desired therapeutic orprophylactic effect is maintained. If symptoms have been alleviated toan appropriate level, treatment may cease. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof symptoms. Patients may also require chronic treatment on a long-termbasis.

C. Lymphodepleting Treatment

In some aspects, the provided methods can further include administeringone or more lymphodepleting therapies, such as prior to or simultaneouswith initiation of administration of the immunotherapy, such as a T celltherapy (e.g. CAR-expressing T cells) or a T cell-engaging therapy. Insome embodiments, the lymphodepleting therapy comprises administrationof a phosphamide, such as cyclophosphamide. In some embodiments, thelymphodepleting therapy can include administration of fludarabine.

In some aspects, preconditioning subjects with immunodepleting (e.g.,lymphodepleting) therapies can improve the effects of adoptive celltherapy (ACT). Preconditioning with lymphodepleting agents, includingcombinations of cyclosporine and fludarabine, have been effective inimproving the efficacy of transferred tumor infiltrating lymphocyte(TIL) cells in cell therapy, including to improve response and/orpersistence of the transferred cells. See, e.g., Dudley et al., Science,298, 850-54 (2002); Rosenberg et al., Clin Cancer Res, 17(13):4550-4557(2011). Likewise, in the context of CAR+ T cells, several studies haveincorporated lymphodepleting agents, most commonly cyclophosphamide,fludarabine, bendamustine, or combinations thereof, sometimesaccompanied by low-dose irradiation. See Han et al. Journal ofHematology & Oncology, 6:47 (2013); Kochenderfer et al., Blood, 119:2709-2720 (2012); Kalos et al., Sci Transl Med, 3(95):95ra73 (2011);Clinical Trial Study Record Nos.: NCT02315612; NCT01822652.

Such preconditioning can be carried out with the goal of reducing therisk of one or more of various outcomes that could dampen efficacy ofthe therapy. These include the phenomenon known as “cytokine sink,” bywhich T cells, B cells, NK cells compete with TILs for homeostatic andactivating cytokines, such as IL-2, IL-7, and/or IL-15; suppression ofTILs by regulatory T cells, NK cells, or other cells of the immunesystem; impact of negative regulators in the tumor microenvironment.Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).

Thus in some embodiments, the provided method further involvesadministering a lymphodepleting therapy to the subject. In someembodiments, the method involves administering the lymphodepletingtherapy to the subject prior to the administration of the dose of cells.In some embodiments, the lymphodepleting therapy contains achemotherapeutic agent such as fludarabine and/or cyclophosphamide. Insome embodiments, the administration of the cells and/or thelymphodepleting therapy is carried out via outpatient delivery.

In some embodiments, the methods include administering a preconditioningagent, such as a lymphodepleting or chemotherapeutic agent, such ascyclophosphamide, fludarabine, or combinations thereof, to a subjectprior to the administration of the dose of cells. For example, thesubject may be administered a preconditioning agent at least 2 daysprior, such as at least 3, 4, 5, 6, or 7 days prior, to the first orsubsequent dose. In some embodiments, the subject is administered apreconditioning agent no more than 7 days prior, such as no more than 6,5, 4, 3, or 2 days prior, to the administration of the dose of cells.

In some embodiments, the subject is preconditioned with cyclophosphamideat a dose between or between about 20 mg/kg and 100 mg/kg, such asbetween or between about 40 mg/kg and 80 mg/kg. In some aspects, thesubject is preconditioned with or with about 60 mg/kg ofcyclophosphamide. In some embodiments, the fludarabine can beadministered in a single dose or can be administered in a plurality ofdoses, such as given daily, every other day or every three days. In someembodiments, the cyclophosphamide is administered once daily for one ortwo days.

In some embodiments, where the lymphodepleting agent comprisesfludarabine, the subject is administered fludarabine at a dose betweenor between about 1 mg/m² and 100 mg/m², such as between or between about10 mg/m² and 75 mg/m², 15 mg/m² and 50 mg/m², 20 mg/m² and 30 mg/m², or24 mg/m² and 26 mg/m². In some instances, the subject is administered 25mg/m² of fludarabine. In some embodiments, the fludarabine can beadministered in a single dose or can be administered in a plurality ofdoses, such as given daily, every other day or every three days. In someembodiments, fludarabine is administered daily, such as for 1-5 days,for example, for 3 to 5 days.

In some embodiments, the lymphodepleting agent comprises a combinationof agents, such as a combination of cyclophosphamide and fludarabine.Thus, the combination of agents may include cyclophosphamide at any doseor administration schedule, such as those described above, andfludarabine at any dose or administration schedule, such as thosedescribed above. For example, in some aspects, the subject isadministered 60 mg/kg (˜2 g/m²) of cyclophosphamide and 3 to 5 doses of25 mg/m² fludarabine prior to the dose of cells.

In one exemplary dosage regime, prior to receiving the first dose,subjects receive a kinase inhibitor 1 day before the administration ofcells and an lymphodepleting preconditioning chemotherapy ofcyclophosphamide and fludarabine (CY/FLU), which is administered atleast two days before the first dose of CAR-expressing cells andgenerally no more than 7 days before administration of cells. In somecases, for example, cyclophosphadmide is given from 24 to 27 days afterthe administration of the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4. After preconditioning treatment, subjectsare administered the dose of CAR-expressing T cells as described above.

In some embodiments, the administration of the preconditioning agentprior to infusion of the dose of cells improves an outcome of thetreatment. For example, in some aspects, preconditioning improves theefficacy of treatment with the dose or increases the persistence of therecombinant receptor-expressing cells (e.g., CAR-expressing cells, suchas CAR-expressing T cells) in the subject. In some embodiments,preconditioning treatment increases disease-free survival, such as thepercent of subjects that are alive and exhibit no minimal residual ormolecularly detectable disease after a given period of time followingthe dose of cells. In some embodiments, the time to median disease-freesurvival is increased.

Once the cells are administered to the subject (e.g., human), thebiological activity of the engineered cell populations in some aspectsis measured by any of a number of known methods. Parameters to assessinclude specific binding of an engineered or natural T cell or otherimmune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., byELISA or flow cytometry. In certain embodiments, the ability of theengineered cells to destroy target cells can be measured using anysuitable method known in the art, such as cytotoxicity assays describedin, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702(2009), and Herman et al. J. Immunological Methods, 285(1): 25-40(2004). In certain embodiments, the biological activity of the cellsalso can be measured by assaying expression and/or secretion of certaincytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects thebiological activity is measured by assessing clinical outcome, such asreduction in tumor burden or load. In some aspects, toxic outcomes,persistence and/or expansion of the cells, and/or presence or absence ofa host immune response, are assessed.

In some embodiments, the administration of the preconditioning agentprior to infusion of the dose of cells improves an outcome of thetreatment such as by improving the efficacy of treatment with the doseor increases the persistence of the recombinant receptor-expressingcells (e.g., CAR-expressing cells, such as CAR-expressing T cells) inthe subject. Therefore, in some embodiments, the dose of preconditioningagent given in the method which is a combination therapy with theinhibitor and cell therapy is higher than the dose given in the methodwithout the inhibitor.

III. T Cell Therapy and Engineering Cells

In some embodiments, the T cell therapy for use in accord with theprovided combination therapy methods includes administering engineeredcells expressing recombinant receptors designed to recognize and/orspecifically bind to molecules associated with the disease or conditionand result in a response, such as an immune response against suchmolecules upon binding to such molecules. The receptors may includechimeric receptors, e.g., chimeric antigen receptors (CARs), and othertransgenic antigen receptors including transgenic T cell receptors(TCRs).

In some embodiments, the cells contain or are engineered to contain anengineered receptor, e.g., an engineered antigen receptor, such as achimeric antigen receptor (CAR), or a T cell receptor (TCR). Alsoprovided are populations of such cells, compositions containing suchcells and/or enriched for such cells, such as in which cells of acertain type such as T cells or CD8⁺ or CD4⁺ cells are enriched orselected. Among the compositions are pharmaceutical compositions andformulations for administration, such as for adoptive cell therapy. Alsoprovided are therapeutic methods for administering the cells andcompositions to subjects, e.g., patients.

Thus, in some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, gene transfer is accomplished by first stimulating thecells, such as by combining it with a stimulus that induces a responsesuch as proliferation, survival, and/or activation, e.g., as measured byexpression of a cytokine or activation marker, followed by transductionof the activated cells, and expansion in culture to numbers sufficientfor clinical applications.

A. Recombinant Receptors

The cells generally express recombinant receptors, such as antigenreceptors including functional non-TCR antigen receptors, e.g., chimericantigen receptors (CARs), and other antigen-binding receptors such astransgenic T cell receptors (TCRs). Also among the receptors are otherchimeric receptors.

1. Chimeric Antigen Receptors (CARs)

Exemplary antigen receptors, including CARs, and methods for engineeringand introducing such receptors into cells, include those described, forexample, in international patent application publication numbersWO2000/14257, WO2013/126726, WO2012/129514, WO2014/031687,WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent applicationpublication numbers US2002131960, US2013287748, US20130149337, U.S. Pat.Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179,6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and8,479,118, and European patent application number EP2537416, and/orthose described by Sadelain et al., Cancer Discov., 3(4): 388-398(2013); Davila et al., PLoS ONE 8(4): e61338 (2013); Turtle et al.,Curr. Opin. Immunol., 24(5): 633-39 (2012); Wu et al., Cancer, 18(2):160-75 (2012). In some aspects, the antigen receptors include a CAR asdescribed in U.S. Pat. No. 7,446,190, and those described inInternational Patent Application Publication No. WO/2014055668 A1.Examples of the CARs include CARs as disclosed in any of theaforementioned publications, such as WO2014031687, U.S. Pat. Nos.8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190,8,389,282, Kochenderfer et al., Nature Reviews Clinical Oncology, 10,267-276 (2013); Wang et al., J. Immunother. 35(9): 689-701 (2012); andBrentjens et al., Sci TranslMed. 5(177) (2013). See also WO2014031687,U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos.7,446,190, and 8,389,282. The chimeric receptors, such as CARs,generally include an extracellular antigen binding domain, such as aportion of an antibody molecule, generally a variable heavy (VH) chainregion and/or variable light (V_(L)) chain region of the antibody, e.g.,an scFv antibody fragment.

In some embodiments, the antigen targeted by the receptor is apolypeptide. In some embodiments, it is a carbohydrate or othermolecule. In some embodiments, the antigen is selectively expressed oroverexpressed on cells of the disease or condition, e.g., the tumor orpathogenic cells, as compared to normal or non-targeted cells ortissues. In other embodiments, the antigen is expressed on normal cellsand/or is expressed on the engineered cells.

Antigens targeted by the receptors in some embodiments include orphantyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22,mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor,CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3,or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA,IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-celladhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2DLigands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72,VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen,PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2,CD123, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin,such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/ormolecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments, the CAR binds a pathogen-specific antigen. In someembodiments, the CAR is specific for viral antigens (such as HIV, HCV,HBV, etc.), bacterial antigens, and/or parasitic antigens.

In some embodiments, the antibody portion of the recombinant receptor,e.g., CAR, further includes at least a portion of an immunoglobulinconstant region, such as a hinge region, e.g., an IgG4 hinge region,and/or a C_(H)1/C_(L) and/or Fc region. In some embodiments, theconstant region or portion is of a human IgG, such as IgG4 or IgG1. Insome aspects, the portion of the constant region serves as a spacerregion between the antigen-recognition component, e.g., scFv, andtransmembrane domain. The spacer can be of a length that provides forincreased responsiveness of the cell following antigen binding, ascompared to in the absence of the spacer. Exemplary spacers, e.g., hingeregions, include those described in international patent applicationpublication number WO2014/031687. In some examples, the spacer is or isabout 12 amino acids in length or is no more than 12 amino acids inlength. Exemplary spacers include those having at least about 10 to 229amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids,about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids,about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20amino acids, or about 10 to 15 amino acids, and including any integerbetween the endpoints of any of the listed ranges. In some embodiments,a spacer region has about 12 amino acids or less, about 119 amino acidsor less, or about 229 amino acids or less. Exemplary spacers includeIgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4hinge linked to the CH3 domain. Exemplary spacers include, but are notlimited to, those described in Hudecek et al., Clin. Cancer Res.,19:3153 (2013), international patent application publication numberWO2014031687, U.S. Pat. No. 8,822,647 or published app. No.US2014/0271635.

In some embodiments, the constant region or portion is of a human IgG,such as IgG4 or IgG1. In some embodiments, the spacer has the sequenceESKYGPPCPPCP (set forth in SEQ ID NO: 1), and is encoded by the sequenceset forth in SEQ ID NO: 2. In some embodiments, the spacer has thesequence set forth in SEQ ID NO: 3. In some embodiments, the spacer hasthe sequence set forth in SEQ ID NO: 4. In some embodiments, theconstant region or portion is of IgD. In some embodiments, the spacerhas the sequence set forth in SEQ ID NO: 5. In some embodiments, thespacer has a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 1, 3, 4 or 5.

This antigen recognition domain generally is linked to one or moreintracellular signaling components, such as signaling components thatmimic activation through an antigen receptor complex, such as a TCRcomplex, in the case of a CAR, and/or signal via another cell surfacereceptor. Thus, in some embodiments, the antigen-binding component(e.g., antibody) is linked to one or more transmembrane andintracellular signaling domains. In some embodiments, the transmembranedomain is fused to the extracellular domain. In one embodiment, atransmembrane domain that naturally is associated with one of thedomains in the receptor, e.g., CAR, is used. In some instances, thetransmembrane domain is selected or modified by amino acid substitutionto avoid binding of such domains to the transmembrane domains of thesame or different surface membrane proteins to minimize interactionswith other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, orCD154. Alternatively the transmembrane domain in some embodiments issynthetic. In some aspects, the synthetic transmembrane domain comprisespredominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain. In some embodiments,the linkage is by linkers, spacers, and/or transmembrane domain(s).

Among the intracellular signaling domains are those that mimic orapproximate a signal through a natural antigen receptor, a signalthrough such a receptor in combination with a costimulatory receptor,and/or a signal through a costimulatory receptor alone. In someembodiments, a short oligo- or polypeptide linker, for example, a linkerof between 2 and 10 amino acids in length, such as one containingglycines and serines, e.g., glycine-serine doublet, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

The receptor, e.g., the CAR, generally includes at least oneintracellular signaling component or components. In some embodiments,the receptor includes an intracellular component of a TCR complex, suchas a TCR CD3 chain that mediates T-cell activation and cytotoxicity,e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portionis linked to one or more cell signaling modules. In some embodiments,cell signaling modules include CD3 transmembrane domain, CD3intracellular signaling domains, and/or other CD transmembrane domains.In some embodiments, the receptor, e.g., CAR, further includes a portionof one or more additional molecules such as Fc receptor γ, CD8, CD4,CD25 or CD16. For example, in some aspects, the CAR or other chimericreceptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fcreceptor 7 and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR or other chimericreceptor, the cytoplasmic domain or intracellular signaling domain ofthe receptor activates at least one of the normal effector functions orresponses of the immune cell, e.g., T cell engineered to express theCAR. For example, in some contexts, the CAR induces a function of a Tcell such as cytolytic activity or T-helper activity, such as secretionof cytokines or other factors. In some embodiments, a truncated portionof an intracellular signaling domain of an antigen receptor component orcostimulatory molecule is used in place of an intact immunostimulatorychain, for example, if it transduces the effector function signal. Insome embodiments, the intracellular signaling domain or domains includethe cytoplasmic sequences of the T cell receptor (TCR), and in someaspects also those of co-receptors that in the natural context act inconcert with such receptors to initiate signal transduction followingantigen receptor engagement.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in some embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. In other embodiments, the CAR does not include a component forgenerating a costimulatory signal. In some aspects, an additional CAR isexpressed in the same cell and provides the component for generating thesecondary or costimulatory signal.

T cell activation is in some aspects described as being mediated by twoclasses of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

In some aspects, the CAR includes a primary cytoplasmic signalingsequence that regulates primary activation of the TCR complex. Primarycytoplasmic signaling sequences that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of ITAM containingprimary cytoplasmic signaling sequences include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22,CD79a, CD79b and CD66d. In some embodiments, cytoplasmic signalingmolecule(s) in the CAR contain(s) a cytoplasmic signaling domain,portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the CAR includes a signaling domain and/ortransmembrane portion of a costimulatory receptor, such as CD28, 4-1BB,OX40, DAP10, and ICOS. In some aspects, the same CAR includes both theactivating and costimulatory components.

In some embodiments, the activating domain is included within one CAR,whereas the costimulatory component is provided by another CARrecognizing another antigen. In some embodiments, the CARs includeactivating or stimulatory CARs, costimulatory CARs, both expressed onthe same cell (see WO2014/055668). In some aspects, the cells includeone or more stimulatory or activating CAR and/or a costimulatory CAR. Insome embodiments, the cells further include inhibitory CARs (iCARs, seeFedorov et al., Sci. Transl. Medicine, 5(215) (2013), such as a CARrecognizing an antigen other than the one associated with and/orspecific for the disease or condition whereby an activating signaldelivered through the disease-targeting CAR is diminished or inhibitedby binding of the inhibitory CAR to its ligand, e.g., to reduceoff-target effects.

In certain embodiments, the intracellular signaling domain comprises aCD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta)intracellular domain. In some embodiments, the intracellular signalingdomain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9)co-stimulatory domains, linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more,costimulatory domains and an activation domain, e.g., primary activationdomain, in the cytoplasmic portion. Exemplary CARs include intracellularcomponents of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the CAR or other antigen receptor further includesa marker and/or cells expressing the CAR or other antigen receptorfurther includes a surrogate marker, such as a cell surface marker,which may be used to confirm transduction or engineering of the cell toexpress the receptor, such as a truncated version of a cell surfacereceptor, such as truncated EGFR (tEGFR). In some aspects, the marker,e.g. surrogate marker, includes all or part (e.g., truncated form) ofCD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In someembodiments, the nucleic acid encoding the marker is operably linked toa polynucleotide encoding for a linker sequence, such as a cleavablelinker sequence, e.g., T2A. For example, a marker, and optionally alinker sequence, can be any as disclosed in PCT Pub. No. WO2014031687.For example, the marker can be a truncated EGFR (tEGFR) that is,optionally, linked to a linker sequence, such as a T2A cleavable linkersequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR)comprises the sequence of amino acids set forth in SEQ ID NO: 7 or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NO: 7. An exemplary T2A linker sequence comprises thesequence of amino acids set forth in SEQ ID NO: 6 or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO: 6.

In some embodiments, the marker is a molecule, e.g., cell surfaceprotein, not naturally found on T cells or not naturally found on thesurface of T cells, or a portion thereof. In some embodiments, themolecule is a non-self molecule, e.g., non-self protein, i.e., one thatis not recognized as “self” by the immune system of the host into whichthe cells will be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/orproduces no effect other than to be used as a marker for geneticengineering, e.g., for selecting cells successfully engineered. In otherembodiments, the marker may be a therapeutic molecule or moleculeotherwise exerting some desired effect, such as a ligand for a cell tobe encountered in vivo, such as a costimulatory or immune checkpointmolecule to enhance and/or dampen responses of the cells upon adoptivetransfer and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or thirdgeneration CARs. In some aspects, a first generation CAR is one thatsolely provides a CD3-chain induced signal upon antigen binding; in someaspects, a second-generation CARs is one that provides such a signal andcostimulatory signal, such as one including an intracellular signalingdomain from a costimulatory receptor such as CD28 or CD137; in someaspects, a third generation CAR is one that includes multiplecostimulatory domains of different costimulatory receptors.

In some embodiments, the chimeric antigen receptor includes anextracellular portion containing an antibody or antibody fragment. Insome aspects, the chimeric antigen receptor includes an extracellularportion containing the antibody or fragment and an intracellularsignaling domain. In some embodiments, the antibody or fragment includesan scFv and the intracellular domain contains an ITAM. In some aspects,the intracellular signaling domain includes a signaling domain of a zetachain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimericantigen receptor includes a transmembrane domain linking theextracellular domain and the intracellular signaling domain. In someaspects, the transmembrane domain contains a transmembrane portion ofCD28. In some embodiments, the chimeric antigen receptor contains anintracellular domain of a T cell costimulatory molecule. Theextracellular domain and transmembrane domain can be linked directly orindirectly. In some embodiments, the extracellular domain andtransmembrane are linked by a spacer, such as any described herein. Insome embodiments, the receptor contains extracellular portion of themolecule from which the transmembrane domain is derived, such as a CD28extracellular portion. In some embodiments, the chimeric antigenreceptor contains an intracellular domain derived from a T cellcostimulatory molecule or a functional variant thereof, such as betweenthe transmembrane domain and intracellular signaling domain. In someaspects, the T cell costimulatory molecule is CD28 or 41BB.

For example, in some embodiments, the CAR contains an antibody, e.g., anantibody fragment, a transmembrane domain that is or contains atransmembrane portion of CD28 or a functional variant thereof, and anintracellular signaling domain containing a signaling portion of CD28 orfunctional variant thereof and a signaling portion of CD3 zeta orfunctional variant thereof. In some embodiments, the CAR contains anantibody, e.g., antibody fragment, a transmembrane domain that is orcontains a transmembrane portion of CD28 or a functional variantthereof, and an intracellular signaling domain containing a signalingportion of a 4-1BB or functional variant thereof and a signaling portionof CD3 zeta or functional variant thereof. In some such embodiments, thereceptor further includes a spacer containing a portion of an Igmolecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4hinge, such as a hinge-only spacer.

In some embodiments, the transmembrane domain of the recombinantreceptor, e.g., the CAR, is or includes a transmembrane domain of humanCD28 (e.g. Accession No. P01747.1) or variant thereof, such as atransmembrane domain that comprises the sequence of amino acids setforth in SEQ ID NO: 8 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 8; in some embodiments,the transmembrane-domain containing portion of the recombinant receptorcomprises the sequence of amino acids set forth in SEQ ID NO: 9 or asequence of amino acids having at least at or about 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto.

In some embodiments, the intracellular signaling component(s) of therecombinant receptor, e.g. the CAR, contains an intracellularcostimulatory signaling domain of human CD28 or a functional variant orportion thereof, such as a domain with an LL to GG substitution atpositions 186-187 of a native CD28 protein. For example, theintracellular signaling domain can comprise the sequence of amino acidsset forth in SEQ ID NO: 10 or 11 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. Insome embodiments, the intracellular domain comprises an intracellularcostimulatory signaling domain of 4-1BB (e.g. Accession No. Q07011.1) orfunctional variant or portion thereof, such as the sequence of aminoacids set forth in SEQ ID NO: 12 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

In some embodiments, the intracellular signaling domain of therecombinant receptor, e.g. the CAR, comprises a human CD3 zetastimulatory signaling domain or functional variant thereof, such as a112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No.7,446,190 or U.S. Pat. No. 8,911,993. For example, in some embodiments,the intracellular signaling domain comprises the sequence of amino acidsas set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14or 15.

In some aspects, the spacer contains only a hinge region of an IgG, suchas only a hinge of IgG4 or IgG1, such as the hinge only spacer set forthin SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ighinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/orCH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., anIgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ IDNO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3.In some embodiments, the spacer is or comprises a glycine-serine richsequence or other flexible linker such as known flexible linkers.

For example, in some embodiments, the CAR includes an antibody such asan antibody fragment, including scFvs, a spacer, such as a spacercontaining a portion of an immunoglobulin molecule, such as a hingeregion and/or one or more constant regions of a heavy chain molecule,such as an Ig-hinge containing spacer, a transmembrane domain containingall or a portion of a CD28-derived transmembrane domain, a CD28-derivedintracellular signaling domain, and a CD3 zeta signaling domain. In someembodiments, the CAR includes an antibody or fragment, such as scFv, aspacer such as any of the Ig-hinge containing spacers, a CD28-derivedtransmembrane domain, a 4-1BB-derived intracellular signaling domain,and a CD3 zeta-derived signaling domain.

In some embodiments, nucleic acid molecules encoding such CAR constructsfurther includes a sequence encoding a T2A ribosomal skip element and/ora tEGFR sequence, e.g., downstream of the sequence encoding the CAR. Insome embodiments, the sequence encodes a T2A ribosomal skip element setforth in SEQ ID NO: 6, or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 6. In some embodiments,T cells expressing an antigen receptor (e.g. CAR) can also be generatedto express a truncated EGFR (EGFRt) as a non-immunogenic selectionepitope (e.g. by introduction of a construct encoding the CAR and EGFRtseparated by a T2A ribosome switch to express two proteins from the sameconstruct), which then can be used as a marker to detect such cells (seee.g. U.S. Pat. No. 8,802,374). In some embodiments, the sequence encodesan tEGFR sequence set forth in SEQ ID NO: 7, or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO: 7.

The recombinant receptors, such as CARs, expressed by the cellsadministered to the subject generally recognize or specifically bind toa molecule that is expressed in, associated with, and/or specific forthe disease or condition or cells thereof being treated. Upon specificbinding to the molecule, e.g., antigen, the receptor generally deliversan immunostimulatory signal, such as an ITAM-transduced signal, into thecell, thereby promoting an immune response targeted to the disease orcondition. For example, in some embodiments, the cells express a CARthat specifically binds to an antigen expressed by a cell or tissue ofthe disease or condition or associated with the disease or condition.

2. TCRs

In some embodiments, engineered cells, such as T cells, are providedthat express a T cell receptor (TCR) or antigen-binding portion thereofthat recognizes an peptide epitope or T cell epitope of a targetpolypeptide, such as an antigen of a tumor, viral or autoimmune protein.

In some embodiments, a “T cell receptor” or “TCR” is a molecule thatcontains a variable α and β chains (also known as TCRα and TCRβ,respectively) or a variable γ and δ chains (also known as TCRα and TCRβ,respectively), or antigen-binding portions thereof, and which is capableof specifically binding to a peptide bound to an MHC molecule. In someembodiments, the TCR is in the αβ form. Typically, TCRs that exist in αβand γδ forms are generally structurally similar, but T cells expressingthem may have distinct anatomical locations or functions. A TCR can befound on the surface of a cell or in soluble form. Generally, a TCR isfound on the surface of T cells (or T lymphocytes) where it is generallyresponsible for recognizing antigens bound to major histocompatibilitycomplex (MHC) molecules.

Unless otherwise stated, the term “TCR” should be understood toencompass full TCRs as well as antigen-binding portions orantigen-binding fragments thereof. In some embodiments, the TCR is anintact or full-length TCR, including TCRs in the ap form or 76 form. Insome embodiments, the TCR is an antigen-binding portion that is lessthan a full-length TCR but that binds to a specific peptide bound in anMHC molecule, such as binds to an MHC-peptide complex. In some cases, anantigen-binding portion or fragment of a TCR can contain only a portionof the structural domains of a full-length or intact TCR, but yet isable to bind the peptide epitope, such as MHC-peptide complex, to whichthe full TCR binds. In some cases, an antigen-binding portion containsthe variable domains of a TCR, such as variable α chain and variable βchain of a TCR, sufficient to form a binding site for binding to aspecific MHC-peptide complex. Generally, the variable chains of a TCRcontain complementarity determining regions involved in recognition ofthe peptide, MHC and/or MHC-peptide complex.

In some embodiments, the variable domains of the TCR containhypervariable loops, or complementarity determining regions (CDRs),which generally are the primary contributors to antigen recognition andbinding capabilities and specificity. In some embodiments, a CDR of aTCR or combination thereof forms all or substantially all of theantigen-binding site of a given TCR molecule. The various CDRs within avariable region of a TCR chain generally are separated by frameworkregions (FRs), which generally display less variability among TCRmolecules as compared to the CDRs (see, e.g., Jores et al., Proc. Nat'lAcad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988;see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In someembodiments, CDR3 is the main CDR responsible for antigen binding orspecificity, or is the most important among the three CDRs on a givenTCR variable region for antigen recognition, and/or for interaction withthe processed peptide portion of the peptide-MHC complex. In somecontexts, the CDR1 of the alpha chain can interact with the N-terminalpart of certain antigenic peptides. In some contexts, CDR1 of the betachain can interact with the C-terminal part of the peptide. In somecontexts, CDR2 contributes most strongly to or is the primary CDRresponsible for the interaction with or recognition of the MHC portionof the MHC-peptide complex. In some embodiments, the variable region ofthe 3-chain can contain a further hypervariable region (CDR4 or HVR4),which generally is involved in superantigen binding and not antigenrecognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

In some embodiments, a TCR also can contain a constant domain, atransmembrane domain and/or a short cytoplasmic tail (see, e.g., Janewayet al., Immunobiology: The Immune System in Health and Disease, 3rd Ed.,Current Biology Publications, p. 4:33, 1997). In some aspects, eachchain of the TCR can possess one N-terminal immunoglobulin variabledomain, one immunoglobulin constant domain, a transmembrane region, anda short cytoplasmic tail at the C-terminal end. In some embodiments, aTCR is associated with invariant proteins of the CD3 complex involved inmediating signal transduction.

In some embodiments, a TCR chain contains one or more constant domain.For example, the extracellular portion of a given TCR chain (e.g.,α-chain or β-chain) can contain two immunoglobulin-like domains, such asa variable domain (e.g., Vα or Vβ; typically amino acids 1 to 116 basedon Kabat numbering Kabat et al., “Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services, Public Health ServiceNational Institutes of Health, 1991, 5th ed.) and a constant domain(e.g., α-chain constant domain or Cα, typically positions 117 to 259 ofthe chain based on Kabat numbering or β chain constant domain or Cβ,typically positions 117 to 295 of the chain based on Kabat) adjacent tothe cell membrane. For example, in some cases, the extracellular portionof the TCR formed by the two chains contains two membrane-proximalconstant domains, and two membrane-distal variable domains, whichvariable domains each contain CDRs. The constant domain of the TCR maycontain short connecting sequences in which a cysteine residue forms adisulfide bond, thereby linking the two chains of the TCR. In someembodiments, a TCR may have an additional cysteine residue in each ofthe α and β chains, such that the TCR contains two disulfide bonds inthe constant domains.

In some embodiments, the TCR chains contain a transmembrane domain. Insome embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chain contains a cytoplasmic tail. In some cases,the structure allows the TCR to associate with other molecules like CD3and subunits thereof. For example, a TCR containing constant domainswith a transmembrane region may anchor the protein in the cell membraneand associate with invariant subunits of the CD3 signaling apparatus orcomplex. The intracellular tails of CD3 signaling subunits (e.g. CD3γ,CD3δ, CD3ε and CD3ζ chains) contain one or more immunoreceptortyrosine-based activation motif or ITAM that are involved in thesignaling capacity of the TCR complex.

In some embodiments, the TCR may be a heterodimer of two chains α and β(or optionally γ and δ) or it may be a single chain TCR construct. Insome embodiments, the TCR is a heterodimer containing two separatechains (α and β chains or γ and δ chains) that are linked, such as by adisulfide bond or disulfide bonds.

In some embodiments, the TCR can be generated from a known TCRsequence(s), such as sequences of Vα,β chains, for which a substantiallyfull-length coding sequence is readily available. Methods for obtainingfull-length TCR sequences, including V chain sequences, from cellsources are well known. In some embodiments, nucleic acids encoding theTCR can be obtained from a variety of sources, such as by polymerasechain reaction (PCR) amplification of TCR-encoding nucleic acids withinor isolated from a given cell or cells, or synthesis of publiclyavailable TCR DNA sequences.

In some embodiments, the TCR is obtained from a biological source, suchas from cells such as from a T cell (e.g. cytotoxic T cell), T-cellhybridomas or other publicly available source. In some embodiments, theT-cells can be obtained from in vivo isolated cells. In someembodiments, the TCR is a thymically selected TCR. In some embodiments,the TCR is a neoepitope-restricted TCR. In some embodiments, the T-cellscan be a cultured T-cell hybridoma or clone. In some embodiments, theTCR or antigen-binding portion thereof can be synthetically generatedfrom knowledge of the sequence of the TCR.

In some embodiments, the TCR is generated from a TCR identified orselected from screening a library of candidate TCRs against a targetpolypeptide antigen, or target T cell epitope thereof. TCR libraries canbe generated by amplification of the repertoire of Vα and Vβ from Tcells isolated from a subject, including cells present in PBMCs, spleenor other lymphoid organ. In some cases, T cells can be amplified fromtumor-infiltrating lymphocytes (TILs). In some embodiments, TCRlibraries can be generated from CD4+ or CD8+ cells. In some embodiments,the TCRs can be amplified from a T cell source of a normal of healthysubject, i.e. normal TCR libraries. In some embodiments, the TCRs can beamplified from a T cell source of a diseased subject, i.e. diseased TCRlibraries. In some embodiments, degenerate primers are used to amplifythe gene repertoire of Vα and Vβ, such as by RT-PCR in samples, such asT cells, obtained from humans. In some embodiments, scTv libraries canbe assembled from naïve Vα and Vβ libraries in which the amplifiedproducts are cloned or assembled to be separated by a linker. Dependingon the source of the subject and cells, the libraries can be HLAallele-specific. Alternatively, in some embodiments, TCR libraries canbe generated by mutagenesis or diversification of a parent or scaffoldTCR molecule. In some aspects, the TCRs are subjected to directedevolution, such as by mutagenesis, e.g., of the α or β chain. In someaspects, particular residues within CDRs of the TCR are altered. In someembodiments, selected TCRs can be modified by affinity maturation. Insome embodiments, antigen-specific T cells may be selected, such as byscreening to assess CTL activity against the peptide. In some aspects,TCRs, e.g. present on the antigen-specific T cells, may be selected,such as by binding activity, e.g., particular affinity or avidity forthe antigen.

In some embodiments, the TCR or antigen-binding portion thereof is onethat has been modified or engineered. In some embodiments, directedevolution methods are used to generate TCRs with altered properties,such as with higher affinity for a specific MHC-peptide complex. In someembodiments, directed evolution is achieved by display methodsincluding, but not limited to, yeast display (Holler et al., (2003) NatImmunol, 4, 55-62; Holler et al., (2000) Proc Natl Acad Sci USA, 97,5387-92), phage display (Li et al., (2005) Nat Biotechnol, 23, 349-54),or T cell display (Chervin et al., (2008) J Immunol Methods, 339,175-84). In some embodiments, display approaches involve engineering, ormodifying, a known, parent or reference TCR. For example, in some cases,a wild-type TCR can be used as a template for producing mutagenized TCRsin which in one or more residues of the CDRs are mutated, and mutantswith an desired altered property, such as higher affinity for a desiredtarget antigen, are selected.

In some embodiments, peptides of a target polypeptide for use inproducing or generating a TCR of interest are known or can be readilyidentified by a skilled artisan. In some embodiments, peptides suitablefor use in generating TCRs or antigen-binding portions can be determinedbased on the presence of an HLA-restricted motif in a target polypeptideof interest, such as a target polypeptide described below. In someembodiments, peptides are identified using computer prediction modelsknown to those of skill in the art. In some embodiments, for predictingMHC class I binding sites, such models include, but are not limited to,ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237, andSYFPEITHI (see Schuler et al., (2007) Immunoinformatics Methods inMolecular Biology, 409(1): 75-93 2007). In some embodiments, theMHC-restricted epitope is HLA-A0201, which is expressed in approximately39-46% of all Caucasians and therefore, represents a suitable choice ofMHC antigen for use preparing a TCR or other MHC-peptide bindingmolecule.

HLA-A0201-binding motifs and the cleavage sites for proteasomes andimmune-proteasomes using computer prediction models are known to thoseof skill in the art. For predicting MHC class I binding sites, suchmodels include, but are not limited to, ProPred1 (described in moredetail in Singh and Raghava, ProPred: prediction of HLA-DR bindingsites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (see Schuleret al., SYFPEITHI, Database for Searching and T-Cell Epitope Prediction.in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-932007)

In some embodiments, the TCR or antigen binding portion thereof may be arecombinantly produced natural protein or mutated form thereof in whichone or more property, such as binding characteristic, has been altered.In some embodiments, a TCR may be derived from one of various animalspecies, such as human, mouse, rat, or other mammal. A TCR may becell-bound or in soluble form. In some embodiments, for purposes of theprovided methods, the TCR is in cell-bound form expressed on the surfaceof a cell.

In some embodiments, the TCR is a full-length TCR. In some embodiments,the TCR is an antigen-binding portion. In some embodiments, the TCR is adimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR(sc-TCR). In some embodiments, a dTCR or scTCR have the structures asdescribed in WO 03/020763, WO 04/033685, WO2011/044186.

In some embodiments, the TCR contains a sequence corresponding to thetransmembrane sequence. In some embodiments, the TCR does contain asequence corresponding to cytoplasmic sequences. In some embodiments,the TCR is capable of forming a TCR complex with CD3. In someembodiments, any of the TCRs, including a dTCR or scTCR, can be linkedto signaling domains that yield an active TCR on the surface of a Tcell. In some embodiments, the TCR is expressed on the surface of cells.

In some embodiments a dTCR contains a first polypeptide wherein asequence corresponding to a TCR α chain variable region sequence isfused to the N terminus of a sequence corresponding to a TCR α chainconstant region extracellular sequence, and a second polypeptide whereina sequence corresponding to a TCR β chain variable region sequence isfused to the N terminus a sequence corresponding to a TCR β chainconstant region extracellular sequence, the first and secondpolypeptides being linked by a disulfide bond. In some embodiments, thebond can correspond to the native inter-chain disulfide bond present innative dimeric αβ TCRs. In some embodiments, the interchain disulfidebonds are not present in a native TCR. For example, in some embodiments,one or more cysteines can be incorporated into the constant regionextracellular sequences of dTCR polypeptide pair. In some cases, both anative and a non-native disulfide bond may be desirable. In someembodiments, the TCR contains a transmembrane sequence to anchor to themembrane.

In some embodiments, a dTCR contains a TCR α chain containing a variableα domain, a constant α domain and a first dimerization motif attached tothe C-terminus of the constant α domain, and a TCR β chain comprising avariable β domain, a constant β domain and a first dimerization motifattached to the C-terminus of the constant β domain, wherein the firstand second dimerization motifs easily interact to form a covalent bondbetween an amino acid in the first dimerization motif and an amino acidin the second dimerization motif linking the TCR α chain and TCR β chaintogether.

In some embodiments, the TCR is a scTCR. Typically, a scTCR can begenerated using methods known to those of skill in the art, See e.g.,Soo Hoo, W. F. et al., PNAS (USA) 89, 4759 (1992); Wülfing, C. andPltückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al., PNAS(USA) 90 3830 (1993); International published PCT Nos. WO 96/13593, WO96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186; andSchlueter, C. J. et al., J. Mol. Biol. 256, 859 (1996). In someembodiments, a scTCR contains an introduced non-native disulfideinterchain bond to facilitate the association of the TCR chains (seee.g. International published PCT No. WO 03/020763). In some embodiments,a scTCR is a non-disulfide linked truncated TCR in which heterologousleucine zippers fused to the C-termini thereof facilitate chainassociation (see e.g. International published PCT No. WO99/60120). Insome embodiments, a scTCR contain a TCRα variable domain covalentlylinked to a TCRβ variable domain via a peptide linker (see e.g.,International published PCT No. WO99/18129).

In some embodiments, a scTCR contains a first segment constituted by anamino acid sequence corresponding to a TCR α chain variable region, asecond segment constituted by an amino acid sequence corresponding to aTCR β chain variable region sequence fused to the N terminus of an aminoacid sequence corresponding to a TCR β chain constant domainextracellular sequence, and a linker sequence linking the C terminus ofthe first segment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by anα chain variable region sequence fused to the N terminus of an α chainextracellular constant domain sequence, and a second segment constitutedby a β chain variable region sequence fused to the N terminus of asequence β chain extracellular constant and transmembrane sequence, and,optionally, a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by aTCR β chain variable region sequence fused to the N terminus of a βchain extracellular constant domain sequence, and a second segmentconstituted by an α chain variable region sequence fused to the Nterminus of a sequence a chain extracellular constant and transmembranesequence, and, optionally, a linker sequence linking the C terminus ofthe first segment to the N terminus of the second segment.

In some embodiments, the linker of a scTCRs that links the first andsecond TCR segments can be any linker capable of forming a singlepolypeptide strand, while retaining TCR binding specificity. In someembodiments, the linker sequence may, for example, have the formula-P-AA-P- wherein P is proline and AA represents an amino acid sequencewherein the amino acids are glycine and serine. In some embodiments, thefirst and second segments are paired so that the variable regionsequences thereof are orientated for such binding. Hence, in some cases,the linker has a sufficient length to span the distance between the Cterminus of the first segment and the N terminus of the second segment,or vice versa, but is not too long to block or reduces bonding of thescTCR to the target ligand. In some embodiments, the linker can containfrom or from about 10 to 45 amino acids, such as 10 to 30 amino acids or26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids.In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P-wherein P is proline, G is glycine and S is serine (SEQ ID NO: 16). Insome embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ IDNO: 17)

In some embodiments, the scTCR contains a covalent disulfide bondlinking a residue of the immunoglobulin region of the constant domain ofthe α chain to a residue of the immunoglobulin region of the constantdomain of the β chain. In some embodiments, the interchain disulfidebond in a native TCR is not present. For example, in some embodiments,one or more cysteines can be incorporated into the constant regionextracellular sequences of the first and second segments of the scTCRpolypeptide. In some cases, both a native and a non-native disulfidebond may be desirable.

In some embodiments of a dTCR or scTCR containing introduced interchaindisulfide bonds, the native disulfide bonds are not present. In someembodiments, the one or more of the native cysteines forming a nativeinterchain disulfide bonds are substituted to another residue, such asto a serine or alanine. In some embodiments, an introduced disulfidebond can be formed by mutating non-cysteine residues on the first andsecond segments to cysteine. Exemplary non-native disulfide bonds of aTCR are described in published International PCT No. WO2006/000830.

In some embodiments, the TCR or antigen-binding fragment thereofexhibits an affinity with an equilibrium binding constant for a targetantigen of between or between about 10⁻⁵ and 10⁻¹² M and all individualvalues and ranges therein. In some embodiments, the target antigen is anMHC-peptide complex or ligand.

In some embodiments, nucleic acid or nucleic acids encoding a TCR, suchas α and β chains, can be amplified by PCR, cloning or other suitablemeans and cloned into a suitable expression vector or vectors. Theexpression vector can be any suitable recombinant expression vector, andcan be used to transform or transfect any suitable host. Suitablevectors include those designed for propagation and expansion or forexpression or both, such as plasmids and viruses.

In some embodiments, the vector can a vector of the pUC series(Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla,Calif), the pET series (Novagen, Madison, Wis.), the pGEX series(Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, PaloAlto, Calif). In some cases, bacteriophage vectors, such as λG10, λGT11,λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. In someembodiments, plant expression vectors can be used and include pBI01,pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some embodiments,animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).In some embodiments, a viral vector is used, such as a retroviralvector.

In some embodiments, the recombinant expression vectors can be preparedusing standard recombinant DNA techniques. In some embodiments, vectorscan contain regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant, or animal) into which the vectoris to be introduced, as appropriate and taking into considerationwhether the vector is DNA- or RNA-based. In some embodiments, the vectorcan contain a nonnative promoter operably linked to the nucleotidesequence encoding the TCR or antigen-binding portion (or otherMHC-peptide binding molecule). In some embodiments, the promoter can bea non-viral promoter or a viral promoter, such as a cytomegalovirus(CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter foundin the long-terminal repeat of the murine stem cell virus. Otherpromoters known to a skilled artisan also are contemplated.

In some embodiments, to generate a vector encoding a TCR, the α and βchains are PCR amplified from total cDNA isolated from a T cell cloneexpressing the TCR of interest and cloned into an expression vector. Insome embodiments, the α and β chains are cloned into the same vector. Insome embodiments, the α and β chains are cloned into different vectors.In some embodiments, the generated α and β chains are incorporated intoa retroviral, e.g. lentiviral, vector.

3. Multi-Targeting

In some embodiments, the cells and methods include multi-targetingstrategies, such as expression of two or more genetically engineeredreceptors on the cell, each recognizing the same of a different antigenand typically each including a different intracellular signalingcomponent. Such multi-targeting strategies are described, for example,in PCT Pub. No. WO 2014055668 A1 (describing combinations of activatingand costimulatory CARs, e.g., targeting two different antigens presentindividually on off-target, e.g., normal cells, but present togetheronly on cells of the disease or condition to be treated) and Fedorov etal., Sci. Transl. Medicine, 5(215) (2013) (describing cells expressingan activating and an inhibitory CAR, such as those in which theactivating CAR binds to one antigen expressed on both normal ornon-diseased cells and cells of the disease or condition to be treated,and the inhibitory CAR binds to another antigen expressed only on thenormal cells or cells which it is not desired to treat).

For example, in some embodiments, the cells include a receptorexpressing a first genetically engineered antigen receptor (e.g., CAR orTCR) which is capable of inducing an activating signal to the cell,generally upon specific binding to the antigen recognized by the firstreceptor, e.g., the first antigen. In some embodiments, the cell furtherincludes a second genetically engineered antigen receptor (e.g., CAR orTCR), e.g., a chimeric costimulatory receptor, which is capable ofinducing a costimulatory signal to the immune cell, generally uponspecific binding to a second antigen recognized by the second receptor.In some embodiments, the first antigen and second antigen are the same.In some embodiments, the first antigen and second antigen are different.

In some embodiments, the first and/or second genetically engineeredantigen receptor (e.g. CAR or TCR) is capable of inducing an activatingsignal to the cell. In some embodiments, the receptor includes anintracellular signaling component containing ITAM or ITAM-like motifs.In some embodiments, the activation induced by the first receptorinvolves a signal transduction or change in protein expression in thecell resulting in initiation of an immune response, such as ITAMphosphorylation and/or initiation of ITAM-mediated signal transductioncascade, formation of an immunological synapse and/or clustering ofmolecules near the bound receptor (e.g. CD4 or CD8, etc.), activation ofone or more transcription factors, such as NF-κB and/or AP-1, and/orinduction of gene expression of factors such as cytokines,proliferation, and/or survival.

In some embodiments, the first and/or second receptor includesintracellular signaling domains of costimulatory receptors such as CD28,CD137 (4-1 BB), OX40, and/or ICOS. In some embodiments, the first andsecond receptors include an intracellular signaling domain of acostimulatory receptor that are different. In one embodiment, the firstreceptor contains a CD28 costimulatory signaling region and the secondreceptor contain a 4-1BB co-stimulatory signaling region or vice versa.

In some embodiments, the first and/or second receptor includes both anintracellular signaling domain containing ITAM or ITAM-like motifs andan intracellular signaling domain of a costimulatory receptor.

In some embodiments, the first receptor contains an intracellularsignaling domain containing ITAM or ITAM-like motifs and the secondreceptor contains an intracellular signaling domain of a costimulatoryreceptor. The costimulatory signal in combination with the activatingsignal induced in the same cell is one that results in an immuneresponse, such as a robust and sustained immune response, such asincreased gene expression, secretion of cytokines and other factors, andT cell mediated effector functions such as cell killing.

In some embodiments, neither ligation of the first receptor alone norligation of the second receptor alone induces a robust immune response.In some aspects, if only one receptor is ligated, the cell becomestolerized or unresponsive to antigen, or inhibited, and/or is notinduced to proliferate or secrete factors or carry out effectorfunctions. In some such embodiments, however, when the plurality ofreceptors are ligated, such as upon encounter of a cell expressing thefirst and second antigens, a desired response is achieved, such as fullimmune activation or stimulation, e.g., as indicated by secretion of oneor more cytokine, proliferation, persistence, and/or carrying out animmune effector function such as cytotoxic killing of a target cell.

In some embodiments, the two receptors induce, respectively, anactivating and an inhibitory signal to the cell, such that binding byone of the receptor to its antigen activates the cell or induces aresponse, but binding by the second inhibitory receptor to its antigeninduces a signal that suppresses or dampens that response. Examples arecombinations of activating CARs and inhibitory CARs or iCARs. Such astrategy may be used, for example, in which the activating CAR binds anantigen expressed in a disease or condition but which is also expressedon normal cells, and the inhibitory receptor binds to a separate antigenwhich is expressed on the normal cells but not cells of the disease orcondition.

In some embodiments, the multi-targeting strategy is employed in a casewhere an antigen associated with a particular disease or condition isexpressed on a non-diseased cell and/or is expressed on the engineeredcell itself, either transiently (e.g., upon stimulation in associationwith genetic engineering) or permanently. In such cases, by requiringligation of two separate and individually specific antigen receptors,specificity, selectivity, and/or efficacy may be improved.

In some embodiments, the plurality of antigens, e.g., the first andsecond antigens, are expressed on the cell, tissue, or disease orcondition being targeted, such as on the cancer cell. In some aspects,the cell, tissue, disease or condition is multiple myeloma or a multiplemyeloma cell. In some embodiments, one or more of the plurality ofantigens generally also is expressed on a cell which it is not desiredto target with the cell therapy, such as a normal or non-diseased cellor tissue, and/or the engineered cells themselves. In such embodiments,by requiring ligation of multiple receptors to achieve a response of thecell, specificity and/or efficacy is achieved.

B. Cells and Preparation of Cells for Genetic Engineering

Among the cells expressing the receptors and administered by theprovided methods are engineered cells. The genetic engineering generallyinvolves introduction of a nucleic acid encoding the recombinant orengineered component into a composition containing the cells, such as byretroviral transduction, transfection, or transformation.

In some embodiments, the nucleic acids are heterologous, i.e., normallynot present in a cell or sample obtained from the cell, such as oneobtained from another organism or cell, which for example, is notordinarily found in the cell being engineered and/or an organism fromwhich such cell is derived. In some embodiments, the nucleic acids arenot naturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

The cells generally are eukaryotic cells, such as mammalian cells, andtypically are human cells. In some embodiments, the cells are derivedfrom the blood, bone marrow, lymph, or lymphoid organs, are cells of theimmune system, such as cells of the innate or adaptive immunity, e.g.,myeloid or lymphoid cells, including lymphocytes, typically T cellsand/or NK cells. Other exemplary cells include stem cells, such asmultipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cells typically are primary cells, such as thoseisolated directly from a subject and/or isolated from a subject andfrozen. In some embodiments, the cells include one or more subsets of Tcells or other cell types, such as whole T cell populations, CD4⁺ cells,CD8⁺ cells, and subpopulations thereof, such as those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. Among the methods includeoff-the-shelf methods. In some aspects, such as for off-the-shelftechnologies, the cells are pluripotent and/or multipotent, such as stemcells, such as induced pluripotent stem cells (iPSCs). In someembodiments, the methods include isolating cells from the subject,preparing, processing, culturing, and/or engineering them, andre-introducing them into the same subject, before or aftercryopreservation.

Among the sub-types and subpopulations of T cells and/or of CD4⁺ and/orof CD8⁺ T cells are naïve T (T_(N)) cells, effector T cells (T_(EFF)),memory T cells and sub-types thereof, such as stem cell memory T(T_(SCM)), central memory T (T_(CM)), effector memory T (T_(EM)), orterminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

In some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, the nucleic acids are heterologous, i.e., normally notpresent in a cell or sample obtained from the cell, such as one obtainedfrom another organism or cell, which for example, is not ordinarilyfound in the cell being engineered and/or an organism from which suchcell is derived. In some embodiments, the nucleic acids are notnaturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

In some embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for introduction of thenucleic acid encoding the transgenic receptor such as the CAR, may beisolated from a sample, such as a biological sample, e.g., one obtainedfrom or derived from a subject. In some embodiments, the subject fromwhich the cell is isolated is one having the disease or condition or inneed of a cell therapy or to which cell therapy will be administered.The subject in some embodiments is a human in need of a particulartherapeutic intervention, such as the adoptive cell therapy for whichcells are being isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig.

In some embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contain cells other thanred blood cells and platelets.

In some embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In some embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In some aspects, a washing step isaccomplished a semi-automated “flow-through” centrifuge (for example,the Cobe 2991 cell processor, Baxter) according to the manufacturer'sinstructions. In some aspects, a washing step is accomplished bytangential flow filtration (TFF) according to the manufacturer'sinstructions. In some embodiments, the cells are resuspended in avariety of biocompatible buffers after washing, such as, for example,Ca⁺⁺/Mg⁺⁺ free PBS. In certain embodiments, components of a blood cellsample are removed and the cells directly resuspended in culture media.

In some embodiments, the methods include density-based cell separationmethods, such as the preparation of white blood cells from peripheralblood by lysing the red blood cells and centrifugation through a Percollor Ficoll gradient.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺, CD4⁺, CD8⁺, CD45RA⁺,and/or CD45RO⁺ T cells, are isolated by positive or negative selectiontechniques.

For example, CD3⁺, CD28⁺ T cells can be positively selected usingCD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 TCell Expander).

In some embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In some embodiments,positive or negative selection is accomplished by incubating cells withone or more antibodies or other binding agent that specifically bind toone or more surface markers expressed or expressed (marker⁺) at arelatively higher level (markerhigh) on the positively or negativelyselected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4⁺ or CD8⁺ selection step is used to separate CD4+ helper and CD8⁺cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8⁺ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (T_(CM)) cells is carriedout to increase efficacy, such as to improve long-term survival,expansion, and/or engraftment following administration, which in someaspects is particularly robust in such sub-populations. See Terakura etal., Blood. 1: 72-82 (2012); Wang et al., J Immunother. 35(9):689-701(2012). In some embodiments, combining TCM-enriched CD8⁺ T cells andCD4⁺ T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L⁺ and CD62L−subsets of CD8⁺ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L-CD8+ and/or CD62L⁺CD8⁺ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (TCM) cells isbased on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD 127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8⁺ population enriched forTCM cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memory T (TCM) cells is carriedout starting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8⁺ cell population or subpopulation, also is used to generate theCD4⁺ cell population or subpopulation, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4⁺ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor CD19, and positive selection based on a marker characteristic ofcentral memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4⁺ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4⁺ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4⁺ T lymphocytes are CD45RO−, CD45RA⁺, CD62L⁺, CD4⁺T cells. In some embodiments, central memory CD4⁺ cells are CD62L⁺ andCD45RO⁺. In some embodiments, effector CD4⁺ cells are CD62L− andCD45RO−.

In one example, to enrich for CD4⁺ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in some embodiments,the cells and cell populations are separated or isolated usingimmunomagnetic (or affinitymagnetic) separation techniques (reviewed inMethods in Molecular Medicine, vol. 58: Metastasis Research Protocols,Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: S. A.Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or microparticles, such asparamagnetic beads (e.g., such as Dynalbeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directlyor indirectly attached to a binding partner, e.g., an antibody, thatspecifically binds to a molecule, e.g., surface marker, present on thecell, cells, or population of cells that it is desired to separate,e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al.,U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies, andmagnetizable particles or antibodies conjugated to cleavable linkers. Insome embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA).Magnetic Activated Cell Sorting (MACS) systems are capable ofhigh-purity selection of cells having magnetized particles attachedthereto. In certain embodiments, MACS operates in a mode wherein thenon-target and target species are sequentially eluted after theapplication of the external magnetic field. That is, the cells attachedto magnetized particles are held in place while the unattached speciesare eluted. Then, after this first elution step is completed, thespecies that were trapped in the magnetic field and were prevented frombeing eluted are freed in some manner such that they can be eluted andrecovered. In certain embodiments, the non-target cells are labelled anddepleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inPCT Pub. Number WO2009/072003, or US 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out usingCliniMACS system (Miltenyi Biotec), for example, for automatedseparation of cells on a clinical-scale level in a closed and sterilesystem. Components can include an integrated microcomputer, magneticseparation unit, peristaltic pump, and various pinch valves. Theintegrated computer in some aspects controls all components of theinstrument and directs the system to perform repeated procedures in astandardized sequence. The magnetic separation unit in some aspectsincludes a movable permanent magnet and a holder for the selectioncolumn. The peristaltic pump controls the flow rate throughout thetubing set and, together with the pinch valves, ensures the controlledflow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses antibody-coupled magnetizableparticles that are supplied in a sterile, non-pyrogenic solution. Insome embodiments, after labelling of cells with magnetic particles thecells are washed to remove excess particles. A cell preparation bag isthen connected to the tubing set, which in turn is connected to a bagcontaining buffer and a cell collection bag. The tubing set consists ofpre-assembled sterile tubing, including a pre-column and a separationcolumn, and are for single use only. After initiation of the separationprogram, the system automatically applies the cell sample onto theseparation column. Labelled cells are retained within the column, whileunlabeled cells are removed by a series of washing steps. In someembodiments, the cell populations for use with the methods describedherein are unlabeled and are not retained in the column. In someembodiments, the cell populations for use with the methods describedherein are labeled and are retained in the column. In some embodiments,the cell populations for use with the methods described herein areeluted from the column after removal of the magnetic field, and arecollected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried outusing the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACSProdigy system in some aspects is equipped with a cell processing unitythat permits automated washing and fractionation of cells bycentrifugation. The CliniMACS Prodigy system can also include an onboardcamera and image recognition software that determines the optimal cellfractionation endpoint by discerning the macroscopic layers of thesource cell product. For example, peripheral blood is automaticallyseparated into erythrocytes, white blood cells and plasma layers. TheCliniMACS Prodigy system can also include an integrated cell cultivationchamber which accomplishes cell culture protocols such as, e.g., celldifferentiation and expansion, antigen loading, and long-term cellculture. Input ports can allow for the sterile removal and replenishmentof media and cells can be monitored using an integrated microscope. See,e.g., Klebanoff et al., J Immunother. 35(9): 651-660 (2012), Terakura etal., Blood. 1:72-82 (2012), and Wang et al., J Immunother. (2012).

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010/033140, Cho et al., Lab Chip 10, 1567-1573 (2010); and Godin etal., J Biophoton. 1(5):355-376 (2008). In both cases, cells can belabeled with multiple markers, allowing for the isolation ofwell-defined T cell subsets at high purity.

In some embodiments, the antibodies or binding partners are labeled withone or more detectable marker, to facilitate separation for positiveand/or negative selection. For example, separation may be based onbinding to fluorescently labeled antibodies. In some examples,separation of cells based on binding of antibodies or other bindingpartners specific for one or more cell surface markers are carried in afluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and/or microelectromechanical systems(MEMS) chips, e.g., in combination with a flow-cytometric detectionsystem. Such methods allow for positive and negative selection based onmultiple markers simultaneously.

In some embodiments, the preparation methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively. The cellsare generally then frozen to −80° C. at a rate of 1° per minute andstored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Theincubation and/or engineering may be carried out in a culture vessel,such as a unit, chamber, well, column, tube, tubing set, valve, vial,culture dish, bag, or other container for culture or cultivating cells.In some embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28,for example, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). In someembodiments, the stimulating agents include IL-2 and/or IL-15, forexample, an IL-2 concentration of at least about 10 units/mL.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al., J Immunother. 35(9): 651-660 (2012), Terakura et al.,Blood. 1:72-82 (2012), and/or Wang et al., J Immunother. 35(9):689-701(2012).

In some embodiments, the T cells are expanded by adding to aculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someaspects, the feeder cells are added to culture medium prior to theaddition of the populations of T cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius. Optionally, the incubation mayfurther comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in someaspects is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4⁺and/or CD8⁺ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

C. Vectors and Methods for Genetic Engineering

Introduction of the nucleic acid molecules encoding the recombinantreceptor may be carried out using any of a number of known vectors. Suchvectors include viral and non-viral systems, including lentiviral andgammaretroviral systems, as well as transposon-based systems such asPiggyBac or Sleeping Beauty-based gene transfer systems. Exemplarymethods include those for transfer of nucleic acids encoding thereceptors, including via viral, e.g., retroviral or lentiviral,transduction, transposons, and electroporation.

In some embodiments, gene transfer is accomplished by first stimulatingthe cell, such as by combining it with a stimulus that induces aresponse such as proliferation, survival, and/or activation, e.g., asmeasured by expression of a cytokine or activation marker, followed bytransduction of the activated cells, and expansion in culture to numberssufficient for clinical applications.

In some embodiments, prior to or during gene transfer, the cells areincubated or cultured in the presence of an inhibitor of a proteintyrosine kinase other than an inhibitor of ITK and/or an inhibitor ofone or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4, including any asdescribed herein. In some embodiments, the inhibitor is added during thecell manufacturing process, for example, during the process ofengineering CAR-T cells. In some aspects, the presence of the inhibitorcan improve the quality of the population of cells produced. In someaspects, the inhibitor of a protein tyrosine kinase other than aninhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 may increase the proliferation orexpansion of cells or may alter one or more signaling pathways therebyresulting in cells with a less-differentiated or less activated surfacephenotype, despite exhibiting substantial expansion and/or effectorfunction.

In some contexts, overexpression of a stimulatory factor (for example, alymphokine or a cytokine) may be toxic to a subject. Thus, in somecontexts, the engineered cells include gene segments that cause thecells to be susceptible to negative selection in vivo, such as uponadministration in adoptive immunotherapy. For example in some aspects,the cells are engineered so that they can be eliminated as a result of achange in the in vivo condition of the patient to which they areadministered. The negative selectable phenotype may result from theinsertion of a gene that confers sensitivity to an administered agent,for example, a compound. Negative selectable genes include the Herpessimplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al.,Cell II:223, 1977) which confers ganciclovir sensitivity; the cellularhypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into T cells using recombinant lentiviral vectorsor retroviral vectors, such as gamma-retroviral vectors (see, e.g.,Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25;Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al.(2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011Nov. 29(11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505.

In some embodiments, recombinant nucleic acids are transferred into Tcells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids aretransferred into T cells via transposition (see, e.g., Manuri et al.(2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec TherNucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic materialin immune cells include calcium phosphate transfection (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.), protoplast fusion, cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the nucleic acids encodingthe recombinant products are those described, e.g., in internationalpatent application, Publication No.: WO2014055668, and U.S. Pat. No.7,446,190.

In some embodiments, the cells, e.g., T cells, may be transfected eitherduring or after expansion e.g. with a T cell receptor (TCR) or achimeric antigen receptor (CAR). This transfection for the introductionof the gene of the desired receptor can be carried out with any suitableretroviral vector, for example. The genetically modified cell populationcan then be liberated from the initial stimulus (the CD3/CD28 stimulus,for example) and subsequently be stimulated with a second type ofstimulus e.g. via a de novo introduced receptor). This second type ofstimulus may include an antigenic stimulus in form of a peptide/MHCmolecule, the cognate (cross-linking) ligand of the geneticallyintroduced receptor (e.g. natural ligand of a CAR) or any ligand (suchas an antibody) that directly binds within the framework of the newreceptor (e.g. by recognizing constant regions within the receptor).See, for example, Cheadle et al, “Chimeric antigen receptors for T-cellbased therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al.,Chimeric Antigen Receptor Therapy for Cancer Annual Review of MedicineVol. 65: 333-347 (2014).

In some cases, a vector may be used that does not require that thecells, e.g., T cells, are activated. In some such instances, the cellsmay be selected and/or transduced prior to activation. Thus, the cellsmay be engineered prior to, or subsequent to culturing of the cells, andin some cases at the same time as or during at least a portion of theculturing.

In some aspects, the cells further are engineered to promote expressionof cytokines or other factors. Among additional nucleic acids, e.g.,genes for introduction are those to improve the efficacy of therapy,such as by promoting viability and/or function of transferred cells;genes to provide a genetic marker for selection and/or evaluation of thecells, such as to assess in vivo survival or localization; genes toimprove safety, for example, by making the cell susceptible to negativeselection in vivo as described by Lupton S. D. et al., Mol. and CellBiol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338(1992); see also the publications of PCT/US91/08442 and PCT/US94/05601by Lupton et al. describing the use of bifunctional selectable fusiongenes derived from fusing a dominant positive selectable marker with anegative selectable marker. See, e.g., Riddell et al., U.S. Pat. No.6,040,177, at columns 14-17.

IV. Exemplary Treatment Outcomes and Methods for Assessing Same

In some embodiments of the methods, compositions, combinations, kits anduses provided herein, the provided combination therapy results in one ormore treatment outcomes, such as a feature associated with any one ormore of the parameters associated with the therapy or treatment, asdescribed below. In some embodiments, the combination therapy canfurther include one or more screening steps to identify subjects fortreatment with the combination therapy and/or continuing the combinationtherapy, and/or a step for assessment of treatment outcomes and/ormonitoring treatment outcomes. In some embodiments, the step forassessment of treatment outcomes can include steps to evaluate and/or tomonitor treatment and/or to identify subjects for administration offurther or remaining steps of the therapy and/or for repeat therapy. Insome embodiments, the screening step and/or assessment of treatmentoutcomes can be used to determine the dose, frequency, duration, timingand/or order of the combination therapy provided herein.

In some embodiments, any of the screening steps and/or assessment oftreatment of outcomes described herein can be used prior to, during,during the course of, or subsequent to administration of one or moresteps of the provided combination therapy, e.g., administration of theimmunotherapy, such as a T cell therapy (e.g. CAR-expressing T cells) ora T cell-engaging therapy, and/or an inhibitor of a protein tyrosinekinase other than an inhibitor of ITK and/or an inhibitor of one or moreof BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4. In some embodiments,assessment is made prior to, during, during the course of, or afterperforming any of the methods provided herein. In some embodiments, theassessment is made prior to performing the methods provided herein. Insome embodiments, assessment is made after performing one or more stepsof the methods provided herein. In some embodiments, the assessment isperformed prior to administration of administration of one or more stepsof the provided combination therapy, for example, to screen and identifypatients suitable and/or susceptible to receive the combination therapy.In some embodiments, the assessment is performed during, during thecourse of, or subsequent to administration of one or more steps of theprovided combination therapy, for example, to assess the intermediate orfinal treatment outcome, e.g., to determine the efficacy of thetreatment and/or to determine whether to continue or repeat thetreatments and/or to determine whether to administer the remaining stepsof the combination therapy.

In some embodiments, treatment of outcomes includes improved immunefunction, e.g., immune function of the T cells administered for cellbased therapy and/or of the endogenous T cells in the body. In someembodiments, exemplary treatment outcomes include, but are not limitedto, enhanced T cell proliferation, enhanced T cell functional activity,changes in immune cell phenotypic marker expression, such as suchfeatures being associated with the engineered T cells, e.g. CAR-T cells,administered to the subject. In some embodiments, exemplary treatmentoutcomes include decreased disease burden, e.g., tumor burden, improvedclinical outcomes and/or enhanced efficacy of therapy.

In some embodiments, the screening step and/or assessment of treatmentof outcomes includes assessing the survival and/or function of the Tcells administered for cell based therapy. In some embodiments, thescreening step and/or assessment of treatment of outcomes includesassessing the levels of cytokines or growth factors. In someembodiments, the screening step and/or assessment of treatment ofoutcomes includes assessing disease burden and/or improvements, e.g.,assessing tumor burden and/or clinical outcomes. In some embodiments,either of the screening step and/or assessment of treatment of outcomescan include any of the assessment methods and/or assays described hereinand/or known in the art, and can be performed one or more times, e.g.,prior to, during, during the course of, or subsequently toadministration of one or more steps of the combination therapy.Exemplary sets of parameters associated with a treatment outcome, whichcan be assessed in some embodiments of the methods provided herein,include peripheral blood immune cell population profile and/or tumorburden.

In some embodiments, the methods affect efficacy of the cell therapy inthe subject. In some embodiments, the persistence, expansion, and/orpresence of recombinant receptor-expressing, e.g., CAR-expressing, cellsin the subject following administration of the dose of cells in themethod with the inhibitor is greater as compared to that achieved via amethod without the administration of the inhibitor. In some embodimentsof the immunotherapy methods provided herein, such as a T cell therapy(e.g. CAR-expressing T cells) or a T cell-engaging therapy, assessmentof the parameter includes assessing the expansion and/or persistence inthe subject of the administered T cells for the immunotherapy, e.g., Tcell therapy, as compared to a method in which the immunotherapy isadministered to the subject in the absence of the inhibitor. In someembodiments, the methods result in the administered T cells exhibitingincreased or prolonged expansion and/or persistence in the subject ascompared to a method in which the T cell therapy is administered to thesubject in the absence of the inhibitor.

In some embodiments, the administration of the inhibitor of a proteintyrosine kinase other than an inhibitor of ITK and/or an inhibitor ofone or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4 decreases diseaseburden, e.g., tumor burden, in the subject as compared to a method inwhich the dose of cells expressing the recombinant receptor isadministered to the subject in the absence of the inhibitor. In someembodiments, the administration of the inhibitor decreases blast marrowin the subject as compared to a method in which the dose of cellsexpressing the recombinant receptor is administered to the subject inthe absence of the inhibitor. In some embodiments, the administration ofthe inhibitor results in improved clinical outcomes, e.g., objectiveresponse rate (ORR), progression-free survival (PFS) and overallsurvival (OS), compared to a method in which the dose of cellsexpressing the recombinant receptor is administered to the subject inthe absence of the inhibitor.

In some embodiments, the subject can be screened prior to theadministration of one or more steps of the combination therapy. Forexample, the subject can be screened for characteristics of the diseaseand/or disease burden, e.g., tumor burden, prior to administration ofthe combination therapy, to determine suitability, responsiveness and/orsusceptibility to administering the combination therapy. In someembodiments, the screening step and/or assessment of treatment outcomescan be used to determine the dose, frequency, duration, timing and/ororder of the combination therapy provided herein.

In some embodiments, the subject can be screened after administration ofone of the steps of the combination therapy, to determine and identifysubjects to receive the remaining steps of the combination therapyand/or to monitor efficacy of the therapy. In some embodiments, thenumber, level or amount of administered T cells and/or proliferationand/or activity of the administered T cells is assessed prior toadministration and/or after administration of the inhibitor.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 is administered until the concentration ornumber of engineered cells in the blood of the subject is (i) at leastat or about 10 engineered cells per microliter, (ii) at least 20%, 30%,40% or 50% of the total number of peripheral blood mononuclear cells(PBMCs), (iii) at least or at least about 1×10⁵ engineered cells; or(iv) at least 5,000 copies of recombinant receptor-encoding DNA permicrograms DNA; and/or at day 90 following the initiation of theadministration in (a), CAR-expressing cells are detectable in the bloodor serum of the subject; and/or at day 90 following the initiation ofthe administration in (a), the blood of the subject contains at least20% CAR-expressing cells, at least 10 CAR-expressing cells permicroliter or at least 1×10⁴ CAR-expressing cells.

In some embodiments, the inhibitor of a protein tyrosine kinase otherthan an inhibitor of ITK and/or an inhibitor of one or more of BTK, TEC,BMX/ETK, RLK/TXK and/or ERBB4 is administered until there is a clinicalbenefit to the treatment, such as at least or greater than a 50%decrease in the total tumor volume a complete response (CR) in whichdetectable tumor has disappeared, progression free survival or diseasefree survival for greater than 6 months or greater than 1 year or more.

In some embodiments, a change and/or an alteration, e.g., an increase,an elevation, a decrease or a reduction, in levels, values ormeasurements of a parameter or outcome compared to the levels, values ormeasurements of the same parameter or outcome in a different time pointof assessment, a different condition, a reference point and/or adifferent subject is determined or assessed. For example, in someembodiments, a fold change, e.g., an increase or decrease, in particularparameters, e.g., number of engineered T cells in a sample, compared tothe same parameter in a different condition, e.g., before or afteradministration of the inhibitor can be determined. In some embodiments,the levels, values or measurements of two or more parameters aredetermined, and relative levels are compared. In some embodiments, thedetermined levels, values or measurements of parameters are compared tothe levels, values or measurements from a control sample or an untreatedsample. In some embodiments, the determined levels, values ormeasurements of parameters are compared to the levels from a sample fromthe same subject but at a different time point. The values obtained inthe quantification of individual parameter can be combined for thepurpose of disease assessment, e.g., by forming an arithmetical orlogical operation on the levels, values or measurements of parameters byusing multi-parametric analysis. In some embodiments, a ratio of two ormore specific parameters can be calculated.

A. T Cell Exposure, Persistence and Proliferation

In some embodiments, the parameter associated with therapy or atreatment outcome, which include parameters that can be assessed for thescreening steps and/or assessment of treatment of outcomes and/ormonitoring treatment outcomes, is or includes assessment of theexposure, persistence and proliferation of the T cells, e.g., T cellsadministered for the T cell based therapy. In some embodiments, theincreased exposure, or prolonged expansion and/or persistence of thecells, and/or changes in cell phenotypes or functional activity of thecells, e.g., cells administered for immunotherapy, e.g. T cell therapy,in the methods provided herein, can be measured by assessing thecharacteristics of the T cells in vitro or ex vivo. In some embodiments,such assays can be used to determine or confirm the function of the Tcells used for the immunotherapy, e.g. T cell therapy, before or afteradministering one or more steps of the combination therapy providedherein.

In some embodiments, the administration of the inhibitor of a proteintyrosine kinase other than an inhibitor of ITK and/or an inhibitor ofone or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4 are designed topromote exposure of the subject to the cells, e.g., T cells administeredfor T cell based therapy, such as by promoting their expansion and/orpersistence over time. In some embodiments, the T cell therapy exhibitsincreased or prolonged expansion and/or persistence in the subject ascompared to a method in which the T cell therapy is administered to thesubject in the absence of the inhibitor.

In some embodiments, the provided methods increase exposure of thesubject to the administered cells (e.g., increased number of cells orduration over time) and/or improve efficacy and therapeutic outcomes ofthe immunotherapy, e.g. T cell therapy. In some aspects, the methods areadvantageous in that a greater and/or longer degree of exposure to thecells expressing the recombinant receptors, e.g., CAR-expressing cells,improves treatment outcomes as compared with other methods. Suchoutcomes may include patient survival and remission, even in individualswith severe tumor burden.

In some embodiments, the administration of the inhibitor of a proteintyrosine kinase other than an inhibitor of ITK and/or an inhibitor ofone or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4 kinase canincrease the maximum, total, and/or duration of exposure to the cells,e.g. T cells administered for the T cell based therapy, in the subjectas compared to administration of the T cells alone in the absence of theinhibitor. In some aspects, administration of the inhibitor, in thecontext of high disease burden (and thus higher amounts of antigen)and/or a more aggressive or resistant cancer enhances efficacy ascompared with administration of the T cells alone in the absence of theinhibitor in the same context, which may result in immunosuppression,anergy and/or exhaustion which may prevent expansion and/or persistenceof the cells.

In some embodiments, the presence and/or amount of cells expressing therecombinant receptor (e.g., CAR-expressing cells administered for T cellbased therapy) in the subject following the administration of the Tcells and before, during and/or after the administration of theinhibitor is detected. In some aspects, quantitative PCR (qPCR) is usedto assess the quantity of cells expressing the recombinant receptor(e.g., CAR-expressing cells administered for T cell based therapy) inthe blood or serum or organ or tissue sample (e.g., disease site, e.g.,tumor sample) of the subject. In some aspects, persistence is quantifiedas copies of DNA or plasmid encoding the receptor, e.g., CAR, permicrogram of DNA, or as the number of receptor-expressing, e.g.,CAR-expressing, cells per microliter of the sample, e.g., of blood orserum, or per total number of peripheral blood mononuclear cells (PBMCs)or white blood cells or T cells per microliter of the sample.

In some embodiments, the cells are detected in the subject at or atleast at 4, 14, 15, 27, or 28 days following the administration of the Tcells, e.g., CAR-expressing T cells. In some aspects, the cells aredetected at or at least at 2, 4, or 6 weeks following, or 3, 6, or 12,18, or 24, or 30 or 36 months, or 1, 2, 3, 4, 5, or more years,following the administration of the T cells, e.g., CAR-expressing Tcells and/or the inhibitor.

In some embodiments, the persistence of receptor-expressing cells (e.g.CAR-expressing cells) in the subject by the methods, following theadministration of the T cells, e.g., CAR-expressing T cells and/or theinhibitor, is greater as compared to that which would be achieved byalternative methods such as those involving the administration of theimmunotherapy alone, e.g., administration the T cells, e.g.,CAR-expressing T cells, in the absence of the inhibitor.

The exposure, e.g., number of cells, e.g. T cells administered for Tcell therapy, indicative of expansion and/or persistence, may be statedin terms of maximum numbers of the cells to which the subject isexposed, duration of detectable cells or cells above a certain number orpercentage, area under the curve for number of cells over time, and/orcombinations thereof and indicators thereof. Such outcomes may beassessed using known methods, such as qPCR to detect copy number ofnucleic acid encoding the recombinant receptor compared to total amountof nucleic acid or DNA in the particular sample, e.g., blood, serum,plasma or tissue, such as a tumor sample, and/or flow cytometric assaysdetecting cells expressing the receptor generally using antibodiesspecific for the receptors. Cell-based assays may also be used to detectthe number or percentage of functional cells, such as cells capable ofbinding to and/or neutralizing and/or inducing responses, e.g.,cytotoxic responses, against cells of the disease or condition orexpressing the antigen recognized by the receptor.

In some aspects, increased exposure of the subject to the cells includesincreased expansion of the cells. In some embodiments, the receptorexpressing cells, e.g. CAR-expressing cells, expand in the subjectfollowing administration of the T cells, e.g., CAR-expressing T cells,and/or following administration of inhibitor. In some aspects, themethods result in greater expansion of the cells compared with othermethods, such as those involving the administration of the T cells,e.g., CAR-expressing T cells, in the absence of administering the tinhibitor.

In some aspects, the method results in high in vivo proliferation of theadministered cells, for example, as measured by flow cytometry. In someaspects, high peak proportions of the cells are detected. For example,in some embodiments, at a peak or maximum level following theadministration of the T cells, e.g., CAR-expressing T cells and/or theinhibitor in the blood or disease-site of the subject or white bloodcell fraction thereof, e.g., PBMC fraction or T cell fraction, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or at least about 90% of the cells express the recombinantreceptor, e.g., the CAR.

In some embodiments, the method results in a maximum concentration, inthe blood or serum or other bodily fluid or organ or tissue of thesubject, of at least 100, 500, 1000, 1500, 2000, 5000, 10,000 or 15,000copies of or nucleic acid encoding the receptor, e.g., the CAR, permicrogram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or0.9 receptor-expressing, e.g., CAR,-expressing cells per total number ofperipheral blood mononuclear cells (PBMCs), total number of mononuclearcells, total number of T cells, or total number of microliters. In someembodiments, the cells expressing the receptor are detected as at least10, 20, 30, 40, 50, or 60% of total PBMCs in the blood of the subject,and/or at such a level for at least 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12,24, 36, 48, or 52 weeks following the T cells, e.g., CAR-expressing Tcells and/or the inhibitor or for 1, 2, 3, 4, or 5, or more yearsfollowing such administration.

In some aspects, the method results in at least a 2-fold, at least a4-fold, at least a 10-fold, or at least a 20-fold increase in copies ofnucleic acid encoding the recombinant receptor, e.g., CAR, per microgramof DNA, e.g., in the serum, plasma, blood or tissue, e.g., tumor sample,of the subject.

In some embodiments, cells expressing the receptor are detectable in theserum, plasma, blood or tissue, e.g., tumor sample, of the subject,e.g., by a specified method, such as qPCR or flow cytometry-baseddetection method, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more days followingadministration of the T cells, e.g., CAR-expressing T cells, or afteradministration of the inhibitor for at least at or about 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24or more weeks following the administration of the T cells, e.g.,CAR-expressing T cells, and/or the inhibitor.

In some aspects, at least about 1×10², at least about 1×10³, at leastabout 1×10⁴, at least about 1×10⁵, or at least about 1×10⁶ or at leastabout 5×10⁶ or at least about 1×10⁷ or at least about 5×10⁷ or at leastabout 1×10⁸ recombinant receptor-expressing, e.g., CAR-expressing cells,and/or at least 10, 25, 50, 100, 200, 300, 400, or 500, or 1000receptor-expressing cells per microliter, e.g., at least 10 permicroliter, are detectable or are present in the subject or fluid,plasma, serum, tissue, or compartment thereof, such as in the blood,e.g., peripheral blood, or disease site, e.g., tumor, thereof. In someembodiments, such a number or concentration of cells is detectable inthe subject for at least about 20 days, at least about 40 days, or atleast about 60 days, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 months, or at least 2 or 3 years, following administration of the Tcells, e.g., CAR-expressing T cells, and/or following the administrationof the inhibitor. Such cell numbers may be as detected by flowcytometry-based or quantitative PCR-based methods and extrapolation tototal cell numbers using known methods. See, e.g., Brentjens et al., SciTransl Med. 2013 5(177), Park et al, Molecular Therapy 15(4):825-833(2007), Savoldo et al., JCI 121(5):1822-1826 (2011), Davila et al.,(2013) PLoS ONE 8(4):e61338, Davila et al., Oncoimmunology1(9):1577-1583 (2012), Lamers, Blood 2011 117:72-82, Jensen et al., BiolBlood Marrow Transplant 2010 September; 16(9): 1245-1256, Brentjens etal., Blood 2011 118(18):4817-4828.

In some aspects, the copy number of nucleic acid encoding therecombinant receptor, e.g., vector copy number, per 100 cells, forexample in the peripheral blood or bone marrow or other compartment, asmeasured by immunohistochemistry, PCR, and/or flow cytometry, is atleast 0.01, at least 0.1, at least 1, or at least 10, at about 1 week,about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at leastabout 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, or 12months or at least 2 or 3 years following administration of the cells,e.g., CAR-expressing T cells, and/or the inhibitor. In some embodiments,the copy number of the vector expressing the receptor, e.g. CAR, permicrogram of genomic DNA is at least 100, at least 1000, at least 5000,or at least 10,000, or at least 15,000 or at least 20,000 at a timeabout 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeksfollowing administration of the T cells, e.g., CAR-expressing T cells,or inhibitor, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 monthsor at least 2 or 3 years following such administration.

In some aspects, the receptor, e.g. CAR, expressed by the cells, isdetectable by quantitative PCR (qPCR) or by flow cytometry in thesubject, plasma, serum, blood, tissue and/or disease site thereof, e.g.,tumor site, at a time that is at least about 3 months, at least about 6months, at least about 12 months, at least about 1 year, at least about2 years, at least about 3 years, or more than 3 years, following theadministration of the cells, e.g., following the initiation of theadministration of the T cells, e.g., CAR-expressing T cells, and/or theinhibitor.

In some embodiments, the area under the curve (AUC) for concentration ofreceptor- (e.g., CAR-) expressing cells in a fluid, plasma, serum,blood, tissue, organ and/or disease site, e.g. tumor site, of thesubject over time following the administration of the T cells, e.g.,CAR-expressing T cells and/or following the administration of theinhibitor, is greater as compared to that achieved via an alternativedosing regimen where the subject is administered the T cells, e.g.,CAR-expressing T cells, in the absence of administering the inhibitor.

In some aspects, the method results in high in vivo proliferation of theadministered cells, for example, as measured by flow cytometry. In someaspects, high peak proportions of the cells are detected. For example,in some embodiments, at a peak or maximum level following the T cells,e.g., CAR-expressing T cells and/or inhibitor, in the blood, plasma,serum, tissue or disease site of the subject or white blood cellfraction thereof, e.g., PBMC fraction or T cell fraction, at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90% of the cells express the recombinantreceptor, e.g., the CAR.

In some aspects, the increased or prolonged expansion and/or persistenceof the dose of cells in the subject administered with the inhibitor isassociated with a benefit in tumor related outcomes in the subject. Insome embodiments, the tumor related outcome includes a decrease in tumorburden or a decrease in blast marrow in the subject. In someembodiments, the tumor burden is decreased by or by at least at or about10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent after administrationof the method. In some embodiments, disease burden, tumor size, tumorvolume, tumor mass, and/or tumor load or bulk is reduced following thedose of cells by at least at or about 50%, 60%, 70%, 80%, 90% or morecompared a subject that has been treated with a method that does notinvolve the administration of a inhibitor of a protein tyrosine kinaseother than an inhibitor of ITK and/or an inhibitor of one or more ofBTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4.

B. T Cell Functional Activity

In some embodiments, parameters associated with therapy or a treatmentoutcome, which include parameters that can be assessed for the screeningsteps and/or assessment of treatment of outcomes and/or monitoringtreatment outcomes, includes one or more of activity, phenotype,proliferation or function of T cells. In some embodiments, any of theknown assays in the art for assessing the activity, phenotypes,proliferation and/or function of the T cells, e.g., T cells administeredfor T cell therapy, can be used. Prior to and/or subsequent toadministration of the cells and/or inhibitor, the biological activity ofthe engineered cell populations in some embodiments is measured, e.g.,by any of a number of known methods. Parameters to assess includespecific binding of an engineered or natural T cell or other immune cellto antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA orflow cytometry. In certain embodiments, the ability of the engineeredcells to destroy target cells can be measured using any suitable methodknown in the art, such as cytotoxicity assays described in, for example,Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Hermanet al., J. Immunological Methods, 285(1): 25-40 (2004). In certainembodiments, the biological activity of the cells is measured byassaying expression and/or secretion of one or more cytokines, such asCD107a, IFNγ, IL-2, GM-CSF and TNFα, and/or by assessing cytolyticactivity.

In some embodiments, assays for the activity, phenotypes, proliferationand/or function of the T cells, e.g., T cells administered for T celltherapy include, but are not limited to, ELISPOT, ELISA, cellularproliferation, cytotoxic lymphocyte (CTL) assay, binding to the T cellepitope, antigen or ligand, or intracellular cytokine staining,proliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. In some embodiments, proliferativeresponses of the T cells can be measured, e.g. by incorporation of³H-thymidine, BrdU (5-Bromo-2′-Deoxyuridine) or2′-deoxy-5-ethynyluridine (EdU) into their DNA or dye dilution assays,using dyes such as carboxyfluorescein diacetate succinimidyl ester(CFSE), CellTrace Violet, or membrane dye PKH26.

In some embodiments, assessing the activity, phenotypes, proliferationand/or function of the T cells, e.g., T cells administered for T celltherapy, include measuring cytokine production from T cells, and/ormeasuring cytokine production in a biological sample from the subject,e.g., plasma, serum, blood, and/or tissue samples, e.g., tumor samples.In some cases, such measured cytokines can include, without limitation,interlekukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4),TNF-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10),interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor(GM-CSF), CD107a, and/or TGF-beta (TGFβ). Assays to measure cytokinesare well known in the art, and include but are not limited to, ELISA,intracellular cytokine staining, cytometric bead array, RT-PCR, ELISPOT,flow cytometry and bio-assays in which cells responsive to the relevantcytokine are tested for responsiveness (e.g. proliferation) in thepresence of a test sample.

In some embodiments, assessing the activity, phenotypes, proliferationand/or function of the T cells, e.g., T cells administered for T celltherapy, include assessing cell phenotypes, e.g., expression ofparticular cell surface markers. In some embodiments, the T cells, e.g.,T cells administered for T cell therapy, are assessed for expression ofT cell activation markers, T cell exhaustion markers, and/or T celldifferentiation markers. In some embodiments, the cell phenotype isassessed before administration. In some embodiments, the cell phenotypeis assessed after administration. T cell activation markers, T cellexhaustion markers, and/or T cell differentiation markers for assessmentinclude any markers known in the art for particular subsets of T cells,e.g., CD25, CD38, human leukocyte antigen-DR (HLA-DR), CD69, CD44,CD137, KLRG1, CD62L^(low), CCR7^(low), CD71, CD2, CD54, CD58, CD244,CD160, programmed cell death protein 1 (PD-1), lymphocyte activationgene 3 protein (LAG-3), T-cell immunoglobulin domain and mucin domainprotein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band Tlymphocyte attenuator (BTLA) and/or T-cell immunoglobulin andimmunoreceptor tyrosine-based inhibitory motif domain (TIGIT) (see,e.g., Liu et al., Cell Death and Disease (2015) 6, e1792). In someembodiments, the assessed cell surface marker is CD25, PD-1 and/orTIM-3. In some embodiments, the assessed cell surface marker is CD25.

In some aspects, detecting the expression levels includes performing anin vitro assay. In some embodiments, the in vitro assay is animmunoassay, an aptamer-based assay, a histological or cytologicalassay, or an mRNA expression level assay. In some embodiments, theparameter or parameters for one or more of each of the one or morefactors, effectors, enzymes and/or surface markers are detected by anenzyme linked immunosorbent assay (ELISA), immunoblotting,immunoprecipitation, radioimmunoassay (RIA), immunostaining, flowcytometry assay, surface plasmon resonance (SPR), chemiluminescenceassay, lateral flow immunoassay, inhibition assay or avidity assay. Insome embodiments, detection of cytokines and/or surface markers isdetermined using a binding reagent that specifically binds to at leastone biomarker. In some cases, the binding reagent is an antibody orantigen-binding fragment thereof, an aptamer or a nucleic acid probe.

In some embodiments, the administration of the inhibitor increases thelevel of circulating CAR T cells. In some embodiments, treatment withthe kinase inhibitor skews the development of T cells towards a Th1immune phenotype. In some embodiments, treatment with ibrutinib or thecompound of Formula (II) may skew CAR T cells towards a more memory-likephenotype that has been associated with increased CAR T in vivopersistence (Busch, D. H., et al. (2016) Semin Immunol, 28(1): 28-34).)

C. Disease Burden, Response, Efficacy and Survival

In some embodiments, parameters associated with therapy or a treatmentoutcome, which include parameters that can be assessed for the screeningsteps and/or assessment of treatment of outcomes and/or monitoringtreatment outcomes, includes tumor or disease burden. The administrationof the immunotherapy, such as a T cell therapy (e.g. CAR-expressing Tcells) or a T cell-engaging therapy and/or the inhibitor, can reduce orprevent the expansion or burden of the disease or condition in thesubject. For example, where the disease or condition is a tumor, themethods generally reduce tumor size, bulk, metastasis, percentage ofblasts in the bone marrow or molecularly detectable cancer and/orimprove prognosis or survival or other symptom associated with tumorburden.

In some embodiments, the provided methods result in a decreased tumorburden in treated subjects compared to alternative methods in which theimmunotherapy, such as a T cell therapy (e.g. CAR-expressing T cells) ora T cell-engaging therapy is given without administration of theinhibitor. It is not necessary that the tumor burden actually be reducedin all subjects receiving the combination therapy, but that tumor burdenis reduced on average in subjects treated, such as based on clinicaldata, in which a majority of subjects treated with such a combinationtherapy exhibit a reduced tumor burden, such as at least 50%, 60%, 70%,80%, 90%, 95% or more of subjects treated with the combination therapy,exhibit a reduced tumor burden.

Disease burden can encompass a total number of cells of the disease inthe subject or in an organ, tissue, or bodily fluid of the subject, suchas the organ or tissue of the tumor or another location, e.g., whichwould indicate metastasis. For example, tumor cells may be detectedand/or quantified in the blood, lymph or bone marrow in the context ofcertain hematological malignancies. Disease burden can include, in someembodiments, the mass of a tumor, the number or extent of metastasesand/or the percentage of blast cells present in the bone marrow.

In some embodiments, the subject has a myeloma, a lymphoma or aleukemia. In some embodiments, the subject has a non-Hodgkin lymphoma(NHL), an acute lymphoblastic leukemia (ALL), a chronic lymphocyticleukemia (CLL), a diffuse large B-cell lymphoma (DLBCL) or a myeloma,e.g., a multiple myeloma (MM). In some embodiments, the subject has a MMor a DBCBL.

In some embodiments, the subject has a solid tumor.

In the case of MM, exemplary parameters to assess the extent of diseaseburden include such parameters as number of clonal plasma cells(e.g., >10% on bone marrow biopsy or in any quantity in a biopsy fromother tissues; plasmacytoma), presence of monoclonal protein(paraprotein) in either serum or urine, evidence of end-organ damagefelt related to the plasma cell disorder (e.g., hypercalcemia (correctedcalcium >2.75 mmol/1); renal insufficiency attributable to myeloma;anemia (hemoglobin <10 g/dl); and/or bone lesions (lytic lesions orosteoporosis with compression fractures)).

In the case of DLBCL, exemplary parameters to assess the extent ofdisease burden include such parameters as cellular morphology (e.g.,centroblastic, immunoblastic, and anaplastic cells), gene expression,miRNA expression and protein expression (e.g., expression of BCL2, BCL6,MUM1, LMO2, MYC, and p21).

In the case of leukemia, the extent of disease burden can be determinedby assessment of residual leukemia in blood or bone marrow. In someembodiments, a subject exhibits morphologic disease if there are greaterthan or equal to 5% blasts in the bone marrow, for example, as detectedby light microscopy. In some embodiments, a subject exhibits complete orclinical remission if there are less than 5% blasts in the bone marrow.

In some embodiments, for leukemia, a subject may exhibit completeremission, but a small proportion of morphologically undetectable (bylight microscopy techniques) residual leukemic cells are present. Asubject is said to exhibit minimum residual disease (MRD) if the subjectexhibits less than 5% blasts in the bone marrow and exhibits molecularlydetectable cancer. In some embodiments, molecularly detectable cancercan be assessed using any of a variety of molecular techniques thatpermit sensitive detection of a small number of cells. In some aspects,such techniques include PCR assays, which can determine unique Ig/T-cellreceptor gene rearrangements or fusion transcripts produced bychromosome translocations. In some embodiments, flow cytometry can beused to identify cancer cell based on leukemia-specificimmunophenotypes. In some embodiments, molecular detection of cancer candetect as few as 1 leukemia cell in 100,000 normal cells. In someembodiments, a subject exhibits MRD that is molecularly detectable if atleast or greater than 1 leukemia cell in 100,000 cells is detected, suchas by PCR or flow cytometry. In some embodiments, the disease burden ofa subject is molecularly undetectable or MRD⁻, such that, in some cases,no leukemia cells are able to be detected in the subject using PCR orflow cytometry techniques.

In some embodiments, the methods and/or administration of animmunotherapy, such as a T cell therapy (e.g. CAR-expressing T cells) ora T cell-engaging therapy and/or inhibitor decrease(s) disease burden ascompared with disease burden at a time immediately prior to theadministration of the immunotherapy, e.g., T cell therapy and/orinhibitor.

In some aspects, administration of the immunotherapy, e.g. T celltherapy and/or inhibitor, may prevent an increase in disease burden, andthis may be evidenced by no change in disease burden.

In some embodiments, the method reduces the burden of the disease orcondition, e.g., number of tumor cells, size of tumor, duration ofpatient survival or event-free survival, to a greater degree and/or fora greater period of time as compared to the reduction that would beobserved with a comparable method using an alternative therapy, such asone in which the subject receives immunotherapy, e.g. T cell therapyalone, in the absence of administration of the inhibitor. In someembodiments, disease burden is reduced to a greater extent or for agreater duration following the combination therapy of administration ofthe immunotherapy, e.g., T cell therapy, and the inhibitor, compared tothe reduction that would be effected by administering each of the agentalone, e.g., administering the inhibitor to a subject having notreceived the immunotherapy, e.g. T cell therapy; or administering theimmunotherapy, e.g. T cell therapy, to a subject having not received theinhibitor.

In some embodiments, the burden of a disease or condition in the subjectis detected, assessed, or measured. Disease burden may be detected insome aspects by detecting the total number of disease ordisease-associated cells, e.g., tumor cells, in the subject, or in anorgan, tissue, or bodily fluid of the subject, such as blood or serum.In some embodiments, disease burden, e.g. tumor burden, is assessed bymeasuring the mass of a solid tumor and/or the number or extent ofmetastases. In some aspects, survival of the subject, survival within acertain time period, extent of survival, presence or duration ofevent-free or symptom-free survival, or relapse-free survival, isassessed. In some embodiments, any symptom of the disease or conditionis assessed. In some embodiments, the measure of disease or conditionburden is specified. In some embodiments, exemplary parameters fordetermination include particular clinical outcomes indicative ofamelioration or improvement in the disease or condition, e.g., tumor.Such parameters include: duration of disease control, including completeresponse (CR), partial response (PR) or stable disease (SD) (see, e.g.,Response Evaluation Criteria In Solid Tumors (RECIST) guidelines),objective response rate (ORR), progression-free survival (PFS) andoverall survival (OS). Specific thresholds for the parameters can be setto determine the efficacy of the method of combination therapy providedherein.

In some aspects, disease burden is measured or detected prior toadministration of the immunotherapy, e.g. T cell therapy, following theadministration of the immunotherapy, e.g. T cell therapy but prior toadministration of the inhibitor following administration of theinhibitor but prior to the administration of the immunotherapy, e.g., Tcell therapy, and/or following the administration of both theimmunotherapy, e.g. T cell therapy and the inhibitor. In the context ofmultiple administration of one or more steps of the combination therapy,disease burden in some embodiments may be measured prior to or followingadministration of any of the steps, doses and/or cycles ofadministration, or at a time between administration of any of the steps,doses and/or cycles of administration.

In some embodiments, the burden is decreased by or by at least at orabout 10, 20, 40, 50, 60, 70, 80, 90, or 100 percent by the providedmethods compared to immediately prior to the administration of theinhibitor and the immunotherapy, e.g. T cell therapy. In someembodiments, disease burden, tumor size, tumor volume, tumor mass,and/or tumor load or bulk is reduced following administration of theimmunotherapy, e.g. T cell therapy and the inhibitor, by at least at orabout 10, 20, 30, 40, 50, 60, 70, 80, 90% or more compared to thatimmediately prior to the administration of the immunotherapy, e.g. Tcell therapy and/or the inhibitor.

In some embodiments, reduction of disease burden by the method comprisesan induction in morphologic complete remission, for example, as assessedat 1 month, 2 months, 3 months, or more than 3 months, afteradministration of, e.g., initiation of, the combination therapy.

In some aspects, an assay for minimal residual disease, for example, asmeasured by multiparametric flow cytometry, is negative, or the level ofminimal residual disease is less than about 0.3%, less than about 0.2%,less than about 0.1%, or less than about 0.05%.

In some embodiments, the event-free survival rate or overall survivalrate of the subject is improved by the methods, as compared with othermethods. For example, in some embodiments, event-free survival rate orprobability for subjects treated by the methods at 6 months followingthe method of combination therapy provided herein, is greater than about40%, greater than about 50%, greater than about 60%, greater than about70%, greater than about 80%, greater than about 90%, or greater thanabout 95%. In some aspects, overall survival rate is greater than about40%, greater than about 50%, greater than about 60%, greater than about70%, greater than about 80%, greater than about 90%, or greater thanabout 95%. In some embodiments, the subject treated with the methodsexhibits event-free survival, relapse-free survival, or survival to atleast 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. Insome embodiments, the time to progression is improved, such as a time toprogression of greater than at or about 6 months, or at least 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 years.

In some embodiments, following treatment by the method, the probabilityof relapse is reduced as compared to other methods. For example, in someembodiments, the probability of relapse at 6 months following the methodof combination therapy, is less than about 80%, less than about 70%,less than about 60%, less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, or less than about 10%.

V. Articles of Manufacture and Kits

Also provided are articles of manufacture containing an inhibitor of aprotein tyrosine kinase other than an inhibitor of ITK and/or aninhibitor of one or more of BTK, TEC, BMX/ETK, RLK/TXK and/or ERBB4,e.g. the compound of Formula (II), and components for the immunotherapy,e.g., antibody or antigen binding fragment thereof or T cell therapy,e.g. engineered cells, and/or compositions thereof. The articles ofmanufacture may include a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, IV solution bags, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer in some embodiments holds a composition which is by itself orcombined with another composition effective for treating, preventingand/or diagnosing the condition. In some embodiments, the container hasa sterile access port. Exemplary containers include an intravenoussolution bags, vials, including those with stoppers pierceable by aneedle for injection, or bottles or vials for orally administeredagents. The label or package insert may indicate that the composition isused for treating a disease or condition.

The article of manufacture may include (a) a first container with acomposition contained therein, wherein the composition includes theantibody or engineered cells used for the immunotherapy, e.g. T celltherapy; and (b) a second container with a composition containedtherein, wherein the composition includes the second agent, such as aninhibitor of a protein tyrosine kinase other than an inhibitor of ITKand/or an inhibitor of one or more of BTK, TEC, BMX/ETK, RLK/TXK and/orERBB4. The article of manufacture may further include a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further include another or the same container comprising apharmaceutically-acceptable buffer. It may further include othermaterials such as other buffers, diluents, filters, needles, and/orsyringes.

VI. Definitions

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

As used herein, a “subject” is a mammal, such as a human or otheranimal, and typically is human. In some embodiments, the subject, e.g.,patient, to whom the immunomodulatory polypeptides, engineered cells, orcompositions are administered, is a mammal, typically a primate, such asa human. In some embodiments, the primate is a monkey or an ape. Thesubject can be male or female and can be any suitable age, includinginfant, juvenile, adolescent, adult, and geriatric subjects. In someembodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to complete or partial amelioration orreduction of a disease or condition or disorder, or a symptom, adverseeffect or outcome, or phenotype associated therewith. Desirable effectsof treatment include, but are not limited to, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.The terms do not imply complete curing of a disease or completeelimination of any symptom or effect(s) on all symptoms or outcomes.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. In some embodiments, the provided cells and compositionsare used to delay development of a disease or to slow the progression ofa disease.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, cells that suppress tumor growthreduce the rate of growth of the tumor compared to the rate of growth ofthe tumor in the absence of the cells.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,cells, or composition, in the context of administration, refers to anamount effective, at dosages/amounts and for periods of time necessary,to achieve a desired result, such as a therapeutic or prophylacticresult.

A “therapeutically effective amount” of an agent, e.g., a pharmaceuticalformulation or engineered cells, refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result, such as for treatment of a disease, condition, ordisorder, and/or pharmacokinetic or pharmacodynamic effect of thetreatment. The therapeutically effective amount may vary according tofactors such as the disease state, age, sex, and weight of the subject,and the immunomodulatory polypeptides or engineered cells administered.In some embodiments, the provided methods involve administering theimmunomodulatory polypeptides, engineered cells, or compositions ateffective amounts, e.g., therapeutically effective amounts.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, recitation that nucleotides or amino acid positions“correspond to” nucleotides or amino acid positions in a disclosedsequence, such as set forth in the Sequence listing, refers tonucleotides or amino acid positions identified upon alignment with thedisclosed sequence to maximize identity using a standard alignmentalgorithm, such as the GAP algorithm. By aligning the sequences, oneskilled in the art can identify corresponding residues, for example,using conserved and identical amino acid residues as guides. In general,to identify corresponding positions, the sequences of amino acids arealigned so that the highest order match is obtained (see, e.g.:Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.” Among thevectors are viral vectors, such as retroviral, e.g., gammaretroviral andlentiviral vectors.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

As used herein, a statement that a cell or population of cells is“positive” for a particular marker refers to the detectable presence onor in the cell of a particular marker, typically a surface marker. Whenreferring to a surface marker, the term refers to the presence ofsurface expression as detected by flow cytometry, for example, bystaining with an antibody that specifically binds to the marker anddetecting said antibody, wherein the staining is detectable by flowcytometry at a level substantially above the staining detected carryingout the same procedure with an isotype-matched control under otherwiseidentical conditions and/or at a level substantially similar to that forcell known to be positive for the marker, and/or at a levelsubstantially higher than that for a cell known to be negative for themarker.

As used herein, a statement that a cell or population of cells is“negative” for a particular marker refers to the absence of substantialdetectable presence on or in the cell of a particular marker, typicallya surface marker. When referring to a surface marker, the term refers tothe absence of surface expression as detected by flow cytometry, forexample, by staining with an antibody that specifically binds to themarker and detecting said antibody, wherein the staining is not detectedby flow cytometry at a level substantially above the staining detectedcarrying out the same procedure with an isotype-matched control underotherwise identical conditions, and/or at a level substantially lowerthan that for cell known to be positive for the marker, and/or at alevel substantially similar as compared to that for a cell known to benegative for the marker.

As used herein, “percent (%) amino acid sequence identity” and “percentidentity” when used with respect to an amino acid sequence (referencepolypeptide sequence) is defined as the percentage of amino acidresidues in a candidate sequence (e.g., the subject antibody orfragment) that are identical with the amino acid residues in thereference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.” It is understood thataspects and variations described herein include “consisting” and/or“consisting essentially of” aspects and variations.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

VII. Exemplary Embodiments

Among the provided embodiments are:

-   -   1. A method of treatment, the method comprising:        -   (a) administering, to a subject having a disease or            condition, T cells that specifically recognize or            specifically bind to an antigen associated with, or            expressed or present on cells of, the disease or condition            and/or a tag comprised by a therapeutic agent that            specifically targets the disease or condition and has been            or is to be administered to the subject; and        -   (b) administering to the subject an inhibitor of a target            protein tyrosine kinase, wherein the inhibitor does not            inhibit ITK and/or inhibits ITK with a half-maximal            inhibitory concentration (IC₅₀) of greater than or greater            than about 1000 nM,        -   wherein the disease or condition (i) is not a B cell-derived            disease or condition (ii) is not associated with expression            of CD19, CD22, or CD20; (iii) does not express the target            protein tyrosine kinase, (iv) does not contain a form of the            target protein tyrosine kinase that is sensitive to the            inhibitor, (v) does not contain a kinase sensitive to the            inhibitor and/or (vi) is not sensitive to inhibition by the            inhibitor and/or wherein the subject or disease or condition            is resistant or refractory to the inhibitor and/or to an            inhibitor of BTK and/or wherein the protein tyrosine kinase            is not ordinarily expressed or is not suspected of being            expressed in cells from which the disease or condition is            derived.    -   2. A method of treatment, the method comprising administering,        to a subject having a disease or condition, T cells that        specifically recognize or specifically bind to an antigen        associated with, or expressed or present on cells of, the        disease or condition and/or a tag comprised by a therapeutic        agent that specifically targets the disease or condition and has        been or is to be administered to the subject, wherein:        -   the subject has been administered an inhibitor of a target            protein tyrosine kinase, wherein the inhibitor does not            inhibit ITK and/or inhibits ITK with a half-maximal            inhibitory concentration (IC₅₀) of greater than or greater            than about 1000 nM; and        -   the disease or condition (i) is not a B cell-derived disease            or condition (ii) is not associated with expression of CD19,            CD22, or CD20; (iii) does not express the protein tyrosine            kinase, (iv) does not contain a form of the target protein            tyrosine kinase that is sensitive to the inhibitor, (v) does            not contain a kinase sensitive to the inhibitor and/or (vi)            is not sensitive to inhibition by the inhibitor and/or            wherein the subject or disease or condition is resistant or            refractory to the inhibitor and/or to an inhibitor of BTK            and/or wherein the protein tyrosine kinase is not ordinarily            expressed or is not suspected of being expressed in cells            from which the disease or condition is derived.    -   3. A method of treatment, the method comprising administering,        to a subject having a disease or condition, an inhibitor of a        target protein tyrosine kinase, wherein the inhibitor does not        inhibit ITK and/or inhibits ITK with a half-maximal inhibitory        concentration (IC₅₀) of greater than or greater than about 1000        nM, wherein:        -   the subject has been administered T cells that specifically            recognize or specifically bind to an antigen associated            with, or expressed or present on cells of, the disease or            condition and/or a tag comprised by a therapeutic agent that            specifically targets the disease or condition and has been            or is to be administered to the subject; and        -   the disease or condition (i) is not a B cell-derived disease            or condition (ii) is not associated with expression of CD19,            CD22, or CD20; (iii) does not express the target protein            tyrosine kinase, (iv) does not contain a form of the target            protein tyrosine kinase that is sensitive to the            inhibitor, (v) does not contain a kinase sensitive to the            inhibitor and/or (vi) is not sensitive to inhibition by the            inhibitor and/or wherein the subject or disease or condition            is resistant or refractory to the inhibitor and/or to an            inhibitor of BTK and/or wherein the TEC family kinase is not            ordinarily expressed or is not suspected of being expressed            in cells from which the disease or condition is derived.    -   4. The method of any of embodiments 1-3, wherein the target        protein tyrosine kinase is tyrosine kinase expressed in        hepatocellular carcinoma (TEC), a resting lymphocyte kinase        (RLK/TXK), a BMX/ETK, or an ERBB4    -   5. The method of any of embodiments 1-4, wherein the target        protein tyrosine kinase is a TEC family kinase.    -   6. The method of any of embodiments 1-5, wherein the inhibitor        is selected from the group consisting of the compound of Formula        (II), ONO/GS-4059, Compound 30 or Compound 38, GDC-0834; RN-486;        CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and        LFM-A13.    -   7. The method of any of embodiments 1-6, wherein:        -   the inhibitor is a selective inhibitor of the target protein            tyrosine kinase; and/or the inhibitor inhibits the target            protein tyrosine kinase with a half-maximal inhibitory            concentration (IC₅₀) that is at least 10 or at least 100            times lower than that of the IC₅₀ of the inhibitor for any            additional protein tyrosine kinase or TEC family kinase,            and/or inhibits the target protein tyrosine kinase with an            IC₅₀ at least 2, at least 10 or at least 100 times lower            than that the IC₅₀ value of the inhibitor for both ITK and            BTK; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) of less            than or less than about 1000 nM, 900 nM, 800 nM, 600 nM, 500            nM, 400 nM, 300 nM, 200 nM, 100 nM or less    -   8. A method of treatment, the method comprising:        -   (1) administering, to a subject having a disease or            condition, T cells that specifically recognize or            specifically bind to an antigen associated with the disease            or condition and/or a tag comprised by a therapeutic agent            that specifically targets the disease or condition and has            been or is to be administered to the subject; and        -   (2) administering to the subject an inhibitor of a target            protein tyrosine kinase, which target protein tyrosine            kinase is a tyrosine kinase expressed in hepatocellular            carcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a            BMX/ETK, or an ERBB4, wherein:        -   the inhibitor is a selective inhibitor of the target protein            tyrosine kinase; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) that is            at least 10 or at least 100 times lower than that of the            IC₅₀ of the inhibitor for any protein tyrosine kinase or TEC            family kinase distinct from the target protein tyrosine            kinase, and/or inhibits the target protein tyrosine kinase            with an IC₅₀ at least 2, at least or at least 100 times            lower than that the IC₅₀ value of the inhibitor for both ITK            and BTK; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) of less            than or less than about 1000 nM, 900 nM, 800 nM, 600 nM, 500            nM, 400 nM, 300 nM, 200 nM, 100 nM or less.    -   9. A method of treatment, the method comprising administering,        to a subject having a disease or condition, T cells that        specifically recognize or specifically bind to an antigen        associated with the disease or condition and/or a tag comprised        by a therapeutic agent that specifically targets the disease or        condition and has been or is to be administered to the subject,        said subject having been administered an inhibitor of a target        protein tyrosine kinase, which target protein tyrosine kinase is        a tyrosine kinase expressed in hepatocellular carcinoma (TEC), a        resting lymphocyte kinase (RLK/TXK), a BMX/ETK, or an ERBB4,        wherein:        -   the inhibitor is a selective inhibitor of the target protein            tyrosine kinase; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) that is            at least 10 or at least 100 times lower than that of the            IC₅₀ of the inhibitor for any protein tyrosine kinase or TEC            family kinase distinct from the target protein tyrosine            kinase, and/or inhibits the target protein tyrosine kinase            with an IC₅₀ at least 2, at least or at least 100 times            lower than that the IC₅₀ value of the inhibitor for both ITK            and BTK; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) of less            than or less than about 1000 nM, 900 nM, 800 nM, 600 nM, 500            nM, 400 nM, 300 nM, 200 nM, 100 nM or less.    -   10. A method of treatment, the method comprising administering        to a subject, having a disease or condition, an inhibitor of a        target protein tyrosine kinase, which target protein tyrosine        kinase is a tyrosine kinase expressed in hepatocellular        carcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a        BMX/ETK, or an ERBB4, said subject having been administered T        cells that specifically recognize or specifically bind to an        antigen associated with the disease or condition and/or a tag        comprised by a therapeutic agent that specifically targets the        disease or condition and has been or is to be administered to        the subject, wherein:        -   the inhibitor is a selective inhibitor of the target protein            tyrosine kinase; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) that is            at least 10 or at least 100 times lower than that of the            IC₅₀ of the inhibitor for any protein tyrosine kinase or TEC            family kinase distinct from the target protein tyrosine            kinase, and/or inhibits the target protein tyrosine kinase            with an IC₅₀ at least 2, at least or at least 100 times            lower than that the IC₅₀ value of the inhibitor for both ITK            and BTK; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) of less            than or less than about 1000 nM, 900 nM, 800 nM, 600 nM, 500            nM, 400 nM, 300 nM, 200 nM, 100 nM or less.    -   11. The method of any of embodiments 8-10, wherein the disease        or condition (i) is not a B cell-derived disease or        condition (ii) is not associated with expression of CD19, CD22,        or CD20; (iii) does not express the target protein tyrosine        kinase, (iv) does not contain a form of the target protein        tyrosine kinase that is sensitive to the inhibitor, (v) does not        contain a kinase sensitive to the inhibitor and/or (vi) is not        sensitive to inhibition by the inhibitor and/or wherein the        subject or disease or condition is resistant or refractory to        the inhibitor and/or to an inhibitor of BTK and/or the target        kinase is not ordinarily expressed or is not suspected of being        expressed in cells from which the disease or condition is        derived.    -   12. The method of any of embodiments 4-11, wherein the target        protein tyrosine kinase is a RLK/TXK.    -   13. The method of any of embodiments 4-12, wherein the target        protein tyrosine kinase is a BMX/ETK and the inhibitor inhibits        Bmx/Etk with an a half-maximal inhibitory concentration (IC₅₀)        that is at least 10 or at least 100 times lower than that of the        IC₅₀ of the inhibitor for any other TEC family kinase and/or for        ITK, and/or inhibits Bmx/Etk with a half-maximal inhibitory        concentration (IC₅₀) of less than or less than about 1000 nM,        900 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM        or less.    -   14. The method of any of embodiments 4 and 7-13, wherein the        target kinase is or comprises an ErbB4.    -   15. The method of any of embodiments 1-13, wherein the inhibitor        comprises is a compound of formula (II):

or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal,polymorph or prodrug thereof

-   -   16. The method of any of embodiments 1-15, wherein the inhibitor        comprises the compound of Formula (II), or an enantiomer,        pharmaceutically-acceptable salt, solvate, hydrate, co-crystal,        polymorph or prodrug thereof or a pharmaceutical composition        comprising any of the foregoing.    -   17. A method of treatment, the method comprising:        -   (1) administering, to a subject having a disease or            condition, T cells comprising a recombinant antigen receptor            that specifically binds to an antigen associated with the            disease or condition and/or a tag comprised by a therapeutic            agent that specifically targets the disease or condition and            has been or is to be administered to the subject; and        -   (2) administering to the subject a kinase inhibitor or a            pharmaceutical composition comprising the inhibitor, wherein            the inhibitor comprises the compound of Formula (II)            comprises the compound of Formula (II), or an enantiomer,            pharmaceutically-acceptable salt, solvate, hydrate,            co-crystal, polymorph or prodrug thereof.    -   18. A method of treatment, the method comprising administering,        to a subject having a disease or condition, T cells comprising a        recombinant antigen receptor that specifically binds to an        antigen associated with the disease or condition and/or a tag        comprised by a therapeutic agent that specifically targets the        disease or condition and has been or is to be administered to        the subject, said subject having been administered a kinase        inhibitor or a pharmaceutical composition comprising the        inhibitor, wherein the inhibitor comprises the compound of        Formula (II) or an enantiomer, pharmaceutically-acceptable salt,        solvate, hydrate, co-crystal, polymorph or prodrug thereof.    -   19. A method of treatment, the method comprising administering,        to a subject having a disease or condition, a kinase inhibitor        or a pharmaceutical composition comprising the inhibitor,        wherein the inhibitor comprises the compound of Formula (II), or        an enantiomer, pharmaceutically-acceptable salt, solvate,        hydrate, co-crystal, polymorph or prodrug thereof, said subject        having been administered T cells comprising a recombinant        antigen receptor that specifically binds to an antigen        associated with the disease or condition and/or a tag comprised        by a therapeutic agent that specifically targets the disease or        condition and has been or is to be administered to the subject.    -   20. The method of any of embodiments 17-19, wherein the disease        or condition is a cancer.    -   21. The method of any of embodiments 1-20, wherein:        -   (i) the subject and/or the disease or condition (a) is            resistant to inhibition of Bruton's tyrosine kinase (BTK)            and/or (b) comprises a population of cells that are            resistant to inhibition by the inhibitor;        -   (ii) the subject and/or the disease or condition comprises a            mutation or disruption in a nucleic acid encoding BTK,            capable of reducing or preventing inhibition of the BTK by            the inhibitor and/or by ibrutinib; and/or        -   (iii) at the time of the administration in (1) and at the            time of the administration in (2) the subject has relapsed            following remission after treatment with, or been deemed            refractory to treatment with the inhibitor and/or with a BTK            inhibitor therapy.    -   22. The method of embodiment 21, wherein the population of cells        is or comprises a population of B cells and/or does not comprise        T cells.    -   23. The method of embodiment 21 or embodiment 22, wherein the        mutation in the nucleic acid encoding BTK comprises a        substitution at position C481, optionally C481S or C481R, and/or        a substitution at position T474, optionally T474I or T474M.    -   24. The method of any of embodiments 1-23, wherein:        -   the target protein tyrosine kinase is not expressed by cells            of the disease or condition, is not ordinarily expressed or            is not suspected of being expressed in cells from which the            disease or condition is derived, and/or        -   the disease or condition is not sensitive to the inhibitor;            and/or at least a plurality of the T cells express the            target protein tyrosine kinase; and/or the target protein            tyrosine kinase is expressed in T cells.    -   25. The method of any of embodiments 1-24, wherein the disease        or condition is a cancer not expressing a B cell antigen, a        non-hematologic cancer, is not a B cell malignancy, is not a B        cell leukemia, or is a solid tumor.    -   26. The method of any of embodiments 1-25, wherein the disease        or condition is a cancer selected from the group consisting of        sarcomas, carcinomas, lymphomas, non-Hodgkin lymphomas (NHLs),        diffuse large B cell lymphoma (DLBCL), leukemia, CLL, ALL, AML        and myeloma.    -   27. The method of any of embodiments 1-26, wherein the disease        or condition is a pancreatic cancer, bladder cancer, colorectal        cancer, breast cancer, prostate cancer, renal cancer,        hepatocellular cancer, lung cancer, ovarian cancer, cervical        cancer, pancreatic cancer, rectal cancer, thyroid cancer,        uterine cancer, gastric cancer, esophageal cancer, head and neck        cancer, melanoma, neuroendocrine cancers, CNS cancers, brain        tumors, bone cancer, or soft tissue sarcoma.    -   28. The method of any of embodiments 1-27, wherein the T cells        recognize or target an antigen selected from ROR1, B cell        maturation antigen (BCMA), tEGFR, Her2, L1-CAM, CD19, CD20,        CD22, mesothelin, CEA, and hepatitis B surface antigen,        anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,        EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR vIII,        FBP, FCRLS, FCRHS, fetal acethycholine e receptor, GD2, GD3,        HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain,        Lewis Y, L1-cell adhesion molecule, (L1-CAM),        Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6,        Preferentially expressed antigen of melanoma (PRAME), survivin,        EGP2, EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9,        GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1,        PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9,        NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6,        dual antigen, and an antigen associated with a universal tag, a        cancer-testes antigen, mesothelin, MUC1, MUC16, PSCA, NKG2D        Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1,        TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate        specific antigen, PSMA, Her2/neu, estrogen receptor,        progesterone receptor, ephrinB2, CD123, c-Met, GD-2,        O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin,        cyclin A2, CCL-1, CD138, and a pathogen-specific antigen.    -   29. The method of any of embodiments 1-28, wherein:        -   the antigen is not a B cell antigen; and/or        -   the antigen is not a B cell antigen selected from the group            consisting of CD19, CD20, CD22, and ROR1.    -   30. The method of embodiment 29, wherein:        -   the antigen is not a B cell antigen selected from the group            consisting of CD19, CD20, CD22, and ROR1; and/or        -   the disease or condition does not express a B cell antigen            selected from the group consisting of CD19, CD20, CD22 and            ROR1 and/or kappa light chain.    -   31. The method of any of embodiments 1-30, wherein the T cells        comprise tumor infiltrating lymphocytes (TILs) or comprises        genetically engineered T cells expressing a recombinant receptor        that specifically binds to the antigen.    -   32. The method of embodiment 31, wherein the T cells comprise        genetically engineered T cells expressing a recombinant receptor        that specifically binds to the antigen or the tag, which        receptor optionally is a chimeric antigen receptor.    -   33. The method of embodiment 31 or embodiment 32, wherein the        recombinant receptor is a transgenic T cell receptor (TCR) or a        functional non-T cell receptor.    -   34. The method of any of embodiments 31-33, wherein the        recombinant receptor is a chimeric receptor, which optionally is        a chimeric antigen receptor (CAR).    -   35. The method of embodiment 34, wherein the chimeric antigen        receptor (CAR) comprises an extracellular antigen-recognition        domain that specifically binds to the antigen and an        intracellular signaling domain comprising an ITAM.    -   36. The method of embodiment 35, wherein the intracellular        signaling domain comprises an intracellular domain of a CD3-zeta        (CD3) chain.    -   37. The method of embodiment 35 or embodiment 36, wherein the        chimeric antigen receptor (CAR) further comprises a        costimulatory signaling region.    -   38. The method of embodiment 37, wherein the costimulatory        signaling region comprises a signaling domain of CD28 or 4-1BB.    -   39. The method of embodiment 37 or embodiment 38, wherein the        costimulatory domain is a domain of CD28.    -   40. The method of any of embodiments 1-39, wherein the inhibitor        is a small molecule, peptide, protein, antibody or        antigen-binding fragment thereof, an antibody mimetic, an        aptamer, or a nucleic acid molecule.    -   41. The method of any of embodiments 1-40, wherein the inhibitor        irreversibly reduces or eliminates the activation of the target        protein tyrosine kinase, specifically binds to a binding site in        the active site of the target protein tyrosine kinase comprising        an amino acid residue corresponding to residue C481 in the        sequence set forth in SEQ ID NO:18, and/or reduces or eliminates        autophosphorylation activity of the target protein tyrosine        kinase.    -   42. The method of any of embodiments 1-41, wherein the inhibitor        is not ibrutinib.    -   43. The method of any of embodiments 1-41, wherein the inhibitor        is not the compound of Formula (II).    -   44. The method of any of embodiments 1-41, wherein the inhibitor        is not GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292;        ONO-4059; CNX-774; and LFM-A13.    -   45. The method of any of embodiments 1, 2-8, 10-17 and 19-44,        wherein the inhibitor is administered concurrently with or        subsequently to initiation of administration of the composition        comprising the T cells.    -   46. The method of any of embodiments 1, 2-8, 10-17 and 19-45,        wherein the inhibitor is administered subsequently to initiation        of administration of the T cells.    -   47. The method of embodiment 45 or embodiment 46, wherein the        inhibitor is administered within, or within about, 1 hour, 2        hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours        or 1 week of the initiation of the administration of the T        cells.    -   48. The method of any of embodiments 45-47, wherein the        inhibitor is administered at a time in which:        -   the number of administered T cells detectable in the blood            from the subject is decreased compared to in the subject at            a preceding time point after initiation of the            administration of the T cells;        -   the number of administered T cells detectable in the blood            is less than or less than about 1.5-fold, 2-fold, 3-fold,            4-fold, 5-fold, 10-fold, 50-fold or 100-fold or less the            peak or maximum number of the cells of the T cell therapy            detectable in the blood of the subject after initiation of            administration of the administration of the T cells; and/or        -   at a time after a peak or maximum level of the administered            T cells are detectable in the blood of the subject, the            number of cells of or derived from the T cells detectable in            the blood from the subject is less than less than 10%, less            than 5%, less than 1% or less than 0.1% of total peripheral            blood mononuclear cells (PBMCs) in the blood of the subject.    -   49. The method of embodiment 48, wherein the increase or        decrease is by greater than or greater than about 1.2-fold,        1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more.    -   50. The method of any of embodiments 1-49, wherein the inhibitor        is administered for a time period up to 2 days, up to 7 days, up        to 14 days, up to 21 days, up to one month, up to two months, up        to three months, up to 6 months or up to 1 year after initiation        of the administration of the administration of the T cells.    -   51. The method of any of embodiments 1, 2-8, 10-17 and 19-50,        wherein the inhibitor is administered up to 3 months after        initiation of the administration of the T cells.    -   52. The method of any of embodiments 1-51, wherein the        administration of the inhibitor is continued, from at least        after initiation of administration of the T cells, until:        -   the number of cells of or derived from the T cells            administered detectable in the blood from the subject is            increased compared to in the subject at a preceding time            point just prior to administration of the inhibitor or            compared to a preceding time point after administration of            the T-cell therapy;        -   the number of cells of or derived from the T cells            detectable in the blood is within 2.0-fold (greater or less)            the peak or maximum number observed in the blood of the            subject after initiation of administration of the T cells;        -   the number of cells of the T cells detectable in the blood            from the subject is greater than or greater than about 10%,            15%, 20%, 30%, 40%, 50%, or 60% total peripheral blood            mononuclear cells (PBMCs) in the blood of the subject;            and/or        -   the subject exhibits a reduction in tumor burden as compared            to tumor burden at a time immediately prior to the            administration of the T cells or at a time immediately prior            to the administration of the inhibitor; and/or        -   the subject exhibits complete or clinical remission.    -   53. The method of any of embodiments 1-52, wherein the inhibitor        is administered orally, subcutaneously or intravenously.    -   54. The method of embodiment 53, wherein the inhibitor is        administered orally.    -   55. The method of any of embodiments 1-54, wherein the inhibitor        is administered six times daily, five times daily, four times        daily, three times daily, twice daily, once daily, every other        day, three times a week or at least once a week.    -   56. The method of embodiment 55, wherein the inhibitor is        administered once daily or twice a day.    -   57. The method of any of embodiments 1-56, wherein the inhibitor        is administered at a total daily dosage amount of at least or at        least about 50 mg/day, 100 mg/day, 150 mg/day, 175 mg/day, 200        mg/day, 250 mg/day, 300 mg/day, 350 mg/day, 400 mg/day, 450        mg/day, 500 mg/day, 600 mg/day, 700 mg/day, 800 mg/day or more.    -   58. The method of any of embodiments 1-57, wherein the inhibitor        is administered in an amount less than or about less than or        about or 420 mg per day.    -   59. The method of any of embodiments 1-58, wherein the        administered T cells comprise T cells that are CD4+ or CD8+.    -   60. The method of any of embodiments 1-59, wherein the        administered T cells comprise cells that are autologous to the        subject.    -   61. The method of any of embodiments 1-60, wherein the        administered T cells comprise T cells that are allogeneic to the        subject.    -   62. The method of any of embodiments 1-61, wherein the        administered T cells comprise administration of a dose        comprising a number of cells between or between about 5×10⁵        cells/kg body weight of the subject and 1×10⁷ cells/kg, 0.5×10⁶        cells/kg and 5×10⁶ cells/kg, between or between about 0.5×10⁶        cells/kg and 3×10⁶ cells/kg, between or between about 0.5×10⁶        cells/kg and 2×10⁶ cells/kg, between or between about 0.5×10⁶        cells/kg and 1×10⁶ cell/kg, between or between about 1.0×10⁶        cells/kg body weight of the subject and 5×10⁶ cells/kg, between        or between about 1.0×10⁶ cells/kg and 3×10⁶ cells/kg, between or        between about 1.0×10⁶ cells/kg and 2×10⁶ cells/kg, between or        between about 2.0×10⁶ cells/kg body weight of the subject and        5×10⁶ cells/kg, between or between about 2.0×10⁶ cells/kg and        3×10⁶ cells/kg, or between or between about 3.0×10⁶ cells/kg        body weight of the subject and 5×10⁶ cells/kg, each inclusive.    -   63. The method of any of embodiments 1-62, wherein the dose of        cells administered is less than the dose in a method in which        the administered T cells are administered without administering        the inhibitor.    -   64. The method of embodiment 63, wherein the dose is at least        1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold less.    -   The method of any of embodiments 1-64, wherein the T cells are        administered in a single dose, which optionally is a single        pharmaceutical composition comprising the cells.    -   66. The method of any of embodiments 1-65, wherein the T cells        are administered as a split dose, wherein the cells of a single        dose are administered in a plurality of compositions,        collectively comprising the cells of the dose, over a period of        no more than three days and/or the method further comprises        administering one or more additional doses of the T cells.    -   67. The method of any of embodiments 1-66, wherein the method        further comprises administering a lymphodepleting chemotherapy        prior to administration of the T cells and/or wherein the        subject has received a lymphodepleting chemotherapy prior to        administration of the T cells.    -   68. The method of embodiment 67, wherein the lymphodepleting        chemotherapy comprises administering fludarabine and/or        cyclophosphamide to the subject.    -   69. The method of any of embodiments 1-68, further comprising:        -   administering an immune modulatory agent to the subject,            wherein the administration of the cells and the            administration of the immune modulatory agent are carried            out simultaneously, separately or in a single composition,            or sequentially, in either order.    -   70. The method of embodiment 69, wherein the immune modulatory        agent is capable of inhibiting or blocking a function of a        molecule, or signaling pathway involving said molecule, wherein        the molecule is an immune-inhibitory molecule and/or wherein the        molecule is an immune checkpoint molecule.    -   71. The method of embodiment 70, wherein the immune checkpoint        molecule or pathway is selected from the group consisting of        PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM3, VISTA, adenosine 2A        Receptor (A2AR), or adenosine or a pathway involving any of the        foregoing.    -   72. The method of any of embodiments 69-71, wherein the immune        modulatory agent is or comprises an antibody, which optionally        is an antibody fragment, a single-chain antibody, a        multispecific antibody, or an immunoconjugate.    -   73. The method of embodiment 72, wherein:        -   the antibody specifically binds to the immune checkpoint            molecule or a ligand or receptor thereof; and/or        -   the antibody is capable of blocking or impairing the            interaction between the immune checkpoint molecule and a            ligand or receptor thereof    -   74. The method of any of embodiments 1-73, wherein the        administered T cells exhibit increased or prolonged expansion        and/or persistence in the subject as compared to a method in        which the administered T cells are administered to the subject        in the absence of the inhibitor.    -   75. The method of any of embodiments 1-74, wherein the method        reduces tumor burden to a greater degree and/or for a greater        period of time as compared to the reduction that would be        observed with a comparable method in which the administered T        cells are administered to the subject in the absence of the        inhibitor.    -   76. A combination, comprising:        -   genetically engineered T cells expressing a recombinant            receptor that binds to an antigen other than a B cell            antigen or other than a B cell antigen selected from the            group consisting of CD19, CD20, CD22 and ROR1, and        -   an inhibitor of a target protein tyrosine kinase, wherein            the inhibitor does not inhibit ITK and/or inhibits ITK with            a half-maximal inhibitory concentration (IC₅₀) of greater            than or greater than about 1000 nM and/or the target protein            tyrosine kinase is a tyrosine kinase expressed in            hepatocellular carcinoma (TEC), a resting lymphocyte kinase            (RLK/TXK), a BMX/ETK, or an ERBB4.    -   77. The combination of embodiment 76, wherein the antigen is        selected from among Her2, L1-CAM, mesothelin, CEA, hepatitis B        surface antigen, anti-folate receptor, CD23, CD24, CD38, CD44,        EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR        vIII, FBP, FCRLS, FCRHS, fetal acethycholine e receptor, GD2,        GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, Lewis Y, L1-cell        adhesion molecule (L1-CAM), Melanoma-associated antigen        (MAGEMAGE-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen        of melanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13        receptor a2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX,        HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a,        CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4,        Fetal AchR, NKG2D ligands, CD44v6, dual antigen, and an antigen        associated with a universal tag, a cancer-testes antigen,        mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1,        gp100, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic        antigen (CEA), prostate specific antigen, PSMA, estrogen        receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2        O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin,        cyclin A2, CCL-1, CD138, and a pathogen-specific antigen.    -   78. The combination of embodiment 76 or embodiment 77, wherein        the antigen is a pathogen-specific antigen, which is a viral        antigen, bacterial antigen or parasitic antigen.    -   79. The combination of any of embodiments 76-78 wherein the        recombinant receptor is a transgenic T cell receptor (TCR) or a        functional non-T cell receptor.    -   80. The combination of any of embodiments 76-79, wherein the        recombinant receptor is a chimeric receptor, which optionally is        a chimeric antigen receptor (CAR).    -   81. The combination of any of embodiments 76-80, wherein:        -   the inhibitor is a selective inhibitor of the target protein            tyrosine kinase; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) that is            at least 10 or at least 100 times lower than that of the            IC₅₀ of the inhibitor for any protein tyrosine kinase or TEC            family kinase distinct from the target protein tyrosine            kinase, and/or inhibits the target protein tyrosine kinase            with an IC₅₀ at least 2, at least or at least 100 times            lower than that the IC₅₀ value of the inhibitor for both ITK            and BTK; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) of less            than or less than about 1000 nM, 900 nM, 800 nM, 600 nM, 500            nM, 400 nM, 300 nM, 200 nM, 100 nM or less.    -   82. The combination of any of embodiments 76-81, wherein the        inhibitor is a small molecule, peptide, protein, antibody or        antigen-binding fragment thereof, an antibody mimetic, an        aptamer, or a nucleic acid molecule.    -   83. The combination of any of embodiments 76-82, wherein the        inhibitor is selected from the group consisting of the compound        of Formula (II), ONO/GS-4059, Compound 30 or Compound 38,        GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;        CNX-774; and LFM-A13.    -   84. The combination of any of embodiments 76-83, wherein the        inhibitor comprises the compound of Formula (II), or an        enantiomer, pharmaceutically-acceptable salt, solvate, hydrate,        co-crystal, polymorph or prodrug thereof or a pharmaceutical        composition comprising any of the foregoing.    -   85. The combination of any of embodiments 76-84 that is        formulated in the same composition.    -   86. The combination of any of embodiments 76-84 that is        formulated in separate compositions.    -   87. A kit, comprising the combination of any of embodiments        76-86 and instructions for administering, to a subject for        treating a disease or condition, optionally a cancer, the        genetically engineered cells and the inhibitor.    -   88. A kit, comprising:        -   a composition comprising a therapeutically effective amount            of genetically engineered T cells expressing a recombinant            receptor that binds to an antigen other than a B cell            antigen or other than a B cell antigen selected from the            group consisting of CD19, CD20, CD22 and ROR1; and        -   instructions for administering, to a subject for treating a            cancer, the genetically engineered cells in a combined            therapy with an inhibitor of a target protein tyrosine            kinase, wherein the inhibitor does not inhibit ITK and/or            inhibits ITK with a half-maximal inhibitory concentration            (IC₅₀) of greater than or greater than about 1000 nM and/or            the target protein tyrosine kinase is a tyrosine kinase            expressed in hepatocellular carcinoma (TEC), a resting            lymphocyte kinase (RLK/TXK), a BMX/ETK, or an ERBB4.    -   89. A kit, comprising:        -   a composition comprising a therapeutically effective amount            of an inhibitor of a target protein tyrosine kinase, wherein            the inhibitor does not inhibit ITK and/or inhibits ITK with            a half-maximal inhibitory concentration (IC₅₀) of greater            than or greater than about 1000 nM and/or the target protein            tyrosine kinase is a tyrosine kinase expressed in            hepatocellular carcinoma (TEC), a resting lymphocyte kinase            (RLK/TXK), a BMX/ETK, or an ERBB4; and        -   instructions for administering, to a subject for treating a            disease or condition, optionally a cancer, the inhibitor in            a combined therapy with genetically engineered T cells, said            T cells expressing a recombinant receptor that binds to an            antigen other than a B cell antigen or other than a B cell            antigen selected from the group consisting of CD19, CD20,            CD22 and ROR1.    -   90. The kit of any of embodiments 87-89, wherein the cancer is        not a cancer expressing a B cell antigen, is a non-hematologic        cancer, is not a B cell malignancy, is not a B cell leukemia, or        is a solid tumor.    -   91. The kit of any of embodiments 87-89, wherein the cancer is a        sarcoma, a carcinoma or a lymphoma, optionally a non-Hodgkin        lymphomas (NHLs), diffuse large B cell lymphoma (DLBCL),        leukemia, CLL, ALL, AML and myeloma.    -   92. The kit of any of embodiments 87-91, wherein the cancer is a        pancreatic cancer, bladder cancer, colorectal cancer, breast        cancer, prostate cancer, renal cancer, hepatocellular cancer,        lung cancer, ovarian cancer, cervical cancer, pancreatic cancer,        rectal cancer, thyroid cancer, uterine cancer, gastric cancer,        esophageal cancer, head and neck cancer, melanoma,        neuroendocrine cancers, CNS cancers, brain tumors, bone cancer,        or soft tissue sarcoma.    -   93. The kit of any of embodiments 87-92, wherein (i) the subject        and/or the disease or condition (a) is resistant to inhibition        of Bruton's tyrosine kinase (BTK) and/or (b) comprises a        population of cells that are resistant to inhibition by the        inhibitor;        -   (ii) the subject and/or the disease or condition comprises a            mutation or disruption in a nucleic acid encoding BTK,            capable of reducing or preventing inhibition of the BTK by            the inhibitor and/or by ibrutinib; and/or        -   (iii) at the time of the administering the subject has            relapsed following remission after treatment with, or been            deemed refractory to treatment with the inhibitor and/or            with a BTK inhibitor therapy.    -   94. The method of embodiment 93, wherein the population of cells        is or comprises a population of B cells and/or does not comprise        T cells.    -   95. The kit of embodiment 93 or embodiment 94, wherein the        mutation in the nucleic acid encoding BTK comprises a        substitution at position C481, optionally C481S or C481R, and/or        a substitution at position T474, optionally T474I or T474M.    -   96. The kit of any of embodiments 87-95, wherein the antigen is        selected from among Her2, L1-CAM, mesothelin, CEA, hepatitis B        surface antigen, anti-folate receptor, CD23, CD24, CD38, CD44,        EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR        vIII, FBP, FCRLS, FCRHS, fetal acethycholine e receptor, GD2,        GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, Lewis Y, L1-cell        adhesion molecule (L1-CAM), Melanoma-associated antigen        (MAGEMAGE-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen        of melanoma (PRAIVIE), survivin, EGP2, EGP40, TAG72, B7-H6,        IL-13 receptor a2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171,        G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate        receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF        receptors, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, and        an antigen associated with a universal tag, a cancer-testes        antigen, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1,        MART-1, gp100, oncofetal antigen, TAG72, VEGF-R2,        carcinoembryonic antigen (CEA), prostate specific antigen, PSMA,        estrogen receptor, progesterone receptor, ephrinB2, CD123,        c-Met, GD-2 O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1),        a cyclin, cyclin A2, CCL-1, CD138, and a pathogen-specific        antigen.    -   97. The kit of any of embodiments 87-96 wherein the antigen is a        pathogen-specific antigen, which is a viral antigen, bacterial        antigen or parasitic antigen.    -   98. The kit of any of embodiments 87-97, wherein the recombinant        receptor is a transgenic T cell receptor (TCR) or a functional        non-T cell receptor.    -   99. The kit of any of embodiments 87-98, wherein the recombinant        receptor is a chimeric receptor, which optionally is a chimeric        antigen receptor (CAR).    -   100. The kit of any of embodiments 87-99, wherein:        -   the inhibitor is a selective inhibitor of the target protein            tyrosine kinase; and/or the inhibitor inhibits the target            protein tyrosine kinase with a half-maximal inhibitory            concentration (IC₅₀) that is at least 10 or at least 100            times lower than that of the IC₅₀ of the inhibitor for any            protein tyrosine kinase or TEC family kinase distinct from            the target protein tyrosine kinase, and/or inhibits the            target protein tyrosine kinase with an IC₅₀ at least 2, at            least or at least 100 times lower than that the IC₅₀ value            of the inhibitor for both ITK and BTK; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) of less            than or less than about 1000 nM, 900 nM, 800 nM, 600 nM, 500            nM, 400 nM, 300 nM, 200 nM, 100 nM or less.    -   101. The kit of any of embodiments 87-100, wherein the inhibitor        is a small molecule, peptide, protein, antibody or        antigen-binding fragment thereof, an antibody mimetic, an        aptamer, or a nucleic acid molecule.    -   102. The kit of any of embodiments 87-101, wherein the inhibitor        is selected from the group consisting of the compound of Formula        (II), ONO/GS-4059, Compound 30 or Compound 38, GDC-0834; RN-486;        CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and        LFM-A13.    -   103. The kit of any of embodiments 87-102, wherein the inhibitor        comprises the compound of Formula (II), or an enantiomer,        pharmaceutically-acceptable salt, solvate, hydrate, co-crystal,        polymorph or prodrug thereof or a pharmaceutical composition        comprising any of the foregoing.    -   104. The kit of any of embodiments 87-103, wherein the        instructions are for administering the inhibitor concurrently        with or subsequently to initiation of administration of the        composition comprising the T cells.    -   105. The kit of any of embodiments 87-104, wherein the        instructions are for administering the inhibitor subsequently to        initiation of administration of the T cells.    -   106. The kit of embodiment 104 or embodiment 105, wherein the        instructions are for administering the inhibitor within, or        within about, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48        hours, 72 hours, 96 hours or 1 week of the initiation of the        administration of the T cells.    -   107. The kit of any of embodiments 104-106, wherein the        instructions are for administering the inhibitor at a time in        which:        -   the number of cells of the T cell therapy detectable in the            blood from the subject is decreased compared to in the            subject at a preceding time point after initiation of the            administration of the T cells;        -   the number of cells of the T cell therapy detectable in the            blood is less than or less than about 1.5-fold, 2-fold,            3-fold, 4-fold, 5-fold, 10-fold, 50-fold or 100-fold or less            the peak or maximum number of the cells of the T cell            therapy detectable in the blood of the subject after            initiation of administration of the administration of the T            cells; and/or        -   at a time after a peak or maximum level of the cells of the            T cell therapy are detectable in the blood of the subject,            the number of cells of or derived from the T cells            detectable in the blood from the subject is less than less            than 10%, less than 5%, less than 1% or less than 0.1% of            total peripheral blood mononuclear cells (PBMCs) in the            blood of the subject.    -   108. The kit of embodiment 107, wherein the increase or decrease        is by greater than or greater than about 1.2-fold, 1.5-fold,        2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more.    -   109. The kit of any of embodiments 87-108, wherein the        instructions are for administering the inhibitor for a time        period up to 2 days, up to 7 days, up to 14 days, up to 21 days,        up to one month, up to two months, up to three months, up to 6        months or up to 1 year after initiation of the administration of        the administration of the T cells.    -   110. The kit of any of embodiments 87-109, wherein the        instructions are for further administering the inhibitor from at        least after initiation of administration of the T cells, until:        -   the number of cells of or derived from the T cells            administered detectable in the blood from the subject is            increased compared to in the subject at a preceding time            point just prior to administration of the inhibitor or            compared to a preceding time point after administration of            the T-cell therapy;        -   the number of cells of or derived from the T cells            detectable in the blood is within 2.0-fold (greater or less)            the peak or maximum number observed in the blood of the            subject after initiation of administration of the T cells;        -   the number of cells of the T cells detectable in the blood            from the subject is greater than or greater than about 10%,            15%, 20%, 30%, 40%, 50%, or 60% total peripheral blood            mononuclear cells (PBMCs) in the blood of the subject;            and/or        -   the subject exhibits a reduction in tumor burden as compared            to tumor burden at a time immediately prior to the            administration of the T cells or at a time immediately prior            to the administration of the inhibitor; and/or        -   the subject exhibits complete or clinical remission.    -   111. The kit of any of embodiments 87-110, wherein the        genetically engineered T cells comprises cells that are        autologous to the subject.    -   112. The kit of any of embodiments 87-111, wherein the        genetically engineered T cells comprises T cells that are        allogeneic to the subject.    -   113. A method of engineering immune cells expressing a        recombinant receptor, comprising:        -   contacting a population of cells comprising T cells with an            inhibitor of a target protein tyrosine kinase, wherein the            inhibitor does not inhibit ITK and/or inhibits ITK with a            half-maximal inhibitory concentration (IC₅₀) of greater than            or greater than about 1000 nM and/or the target protein            tyrosine kinase is a tyrosine kinase expressed in            hepatocellular carcinoma (TEC), a resting lymphocyte kinase            (RLK/TXK), a BMX/ETK, or an ERBB4; and        -   introducing a nucleic acid encoding a recombinant receptor            into the population of T cells under conditions such that            the recombinant receptor is expressed.    -   114. The method of embodiment 113, wherein the population of        cells is or comprises T cells, optionally CD4+ or CD8+.    -   115. The method of embodiment 113 or embodiment 114, wherein the        population of cells are isolated from a subject, optionally a        human subject.    -   116. The method of any of embodiments 113-115, wherein the        contacting occurs prior to and/or during the introducing.    -   117. A method of producing genetically engineered T cells,        comprising introducing a nucleic acid molecule encoding a        recombinant receptor into a primary T cell, wherein the T cells        is from a subject having been administered an inhibitor of a        target protein tyrosine kinase, wherein the inhibitor does not        inhibit ITK and/or inhibits ITK with a half-maximal inhibitory        concentration (IC₅₀) of greater than or greater than about 1000        nM and/or the target protein tyrosine kinase is a tyrosine        kinase expressed in hepatocellular carcinoma (TEC), a resting        lymphocyte kinase (RLK/TXK), a BMX/ETK, or an ERBB4.    -   118. The method of embodiment 117, wherein the subject has been        administered the inhibitor no more than 30 days, 20 days, 10        days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2        days, or 1 day prior to introducing the nucleic acid molecule.    -   119. The method of any of embodiments 113-118, wherein:        -   the inhibitor is a selective inhibitor of the target protein            tyrosine kinase; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) that is            at least 10 or at least 100 times lower than that of the            IC₅₀ of the inhibitor for any protein tyrosine kinase or TEC            family kinase distinct from the target protein tyrosine            kinase, and/or inhibits the target protein tyrosine kinase            with an IC₅₀ at least 2, at least or at least 100 times            lower than that the IC₅₀ value of the inhibitor for both ITK            and BTK; and/or        -   the inhibitor inhibits the target protein tyrosine kinase            with a half-maximal inhibitory concentration (IC₅₀) of less            than or less than about 1000 nM, 900 nM, 800 nM, 600 nM, 500            nM, 400 nM, 300 nM, 200 nM, 100 nM or less.    -   120. The method of any of embodiments 113-119, wherein the        inhibitor is a small molecule, peptide, protein, antibody or        antigen-binding fragment thereof, an antibody mimetic, an        aptamer, or a nucleic acid molecule.    -   121. The method of any of embodiments 113-120, wherein the        inhibitor is selected from the group consisting of the compound        of Formula (II), ONO/GS-4059, Compound 30 or Compound 38,        GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;        CNX-774; and LFM-A13.    -   122. The method of any of embodiments 113-121, wherein the        inhibitor comprises the compound of Formula (II), or an        enantiomer, pharmaceutically-acceptable salt, solvate, hydrate,        co-crystal, polymorph or prodrug thereof or a pharmaceutical        composition comprising any of the foregoing.    -   123. The method of any of embodiments 113-122, wherein the T        cells comprise CD4+ or CD8+ cells.

VIII. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Assessment of CAR-Expressing T Cell Phenotype and Function inthe Presence of an Inhibitor of a TEC Family

Properties of CAR-expressing T cells in the presence of an inhibitor ofa TEC family kinase, ibrutinib or the compound of Formula (II), wereassessed in in vitro studies.

To generate CAR-expressing T cells, T cells were isolated byimmunoaffinity-based enrichment from three healthy human donor subjects,and cells from each donor were activated and transduced with a viralvector encoding an anti-CD19 CAR. The CAR contained an anti-CD19 scFv,an Ig-derived spacer, a human CD28-derived transmembrane domain, a human4-1BB-derived intracellular signaling domain and a human CD3zeta-derived signaling domain. The nucleic acid construct encoding theCAR also included a truncated EGFR (tEGFR) sequence for use as atransduction marker, separated from the CAR sequence by a self-cleavingT2A sequence.

CAR-expressing CD4+ and CD8+ cells were mixed 1:1 for each donor,individually, and the pooled cells for each donor assessed in vitrounder various conditions.

A. Cytolytic Activity

CAR T cells generated as described above were plated in triplicate onPoly-D-Lysine plates and then co-cultured with ibrutinib-resistant,CD19-expressing target cells (K562 cells transduced to express CD19,K562-CD19) at an effector to target (E:T) ratio of 2.5:1. The targetscells were labeled with NucLight Red (NLR) to permit tracking of targetcells by microscopy. Ibrutinib was added to the cultures atconcentrations of 5000, 500, 50, 5 and 0.5 nM (reflecting a dosage rangecovering doses observed to be supraphysiologic (500 nM) and Cmax (227nM)). The compound of Formula (II) was added to the cultures atconcentrations of 5000, 500, 50, 5 and 0.5 nm (reflecting a dosage rangecovering doses observed to be supraphysiologic (5000 nM) and C_(max)(1.8 μM)). CAR-T cells incubated in the presence of target cells in theabsence of the inhibitor were used as an “untreated” control. Cytolyticactivity was assessed by measuring the loss of viable target cells overa period of four days, as determined by red fluorescent signal (usingthe IncuCyte® Live Cell Analysis System, Essen Bioscience). Percent (%)of target killing was assessed by measuring area under the curve (AUC)for normalized target cell count over time and normalizing the inverseAUC (1/AUC) values by defining a 0% value (target cells alone) and a100% value (CAR+ T cells co-cultured with target cells in vehiclecontrol).

As shown by microscopy, after an initial period of target cell growth,anti-CD19 CAR T cells from all donors were able to reduce the targetcell number over a period of four days, thus demonstrating effectivekilling in the assay (FIG. 1A). A representative image of target cellsco-cultured with CAR T cells at the start and end of the cytotoxic assayis shown in FIG. 1B.

Target-specific cytolytic activity by CAR-T cells against target cells,in the presence or absence of a range of doses of ibrutinib or thecompound of Formula (II), was normalized to results for untreatedcontrols. As shown in FIG. 1C, for two donors, ibrutinib did notsignificantly impact cytolytic activity, even when the concentrationswere increased to supra-physiological levels (500 nM). The addition ofibrutinib, at all concentrations tested during the co-culture, did notinhibit the cytolytic function of the anti-CD19 CAR T cells. However, amodestly increased target cell killing was observed for one donortreated with ibrutinib (P <0.0001) (FIG. 1C). FIG. 1D also shows thatsimilar results were observed for cells from each of the same threedonors when treated with the compound of Formula (II) at concentrationsfrom to 5000 nM, a range including a dose observed to besupraphysiologic (5000 nM) and an observed Cmax (1.811M). Thus, bothinhibitors were observed to have no negative impact on cytolyticfunction of CAR-T cells at dose ranges spanning Cmax or supraphysiologicdoses assessed.

B. Expression of CAR-T Cell Surface Makers.

To assess various phenotypic markers of anti-CD19 CART cells cultured inthe presence of ibrutinib or the compound of Formula (II), a panel ofactivation markers on CAR+, CD4+ and CD8+ cells (from three donors) weretracked over 4 days following stimulation with irradiated K562 targetcells expressing the CD19 cognate antigen. CAR-T cells generated asdescribed above were plated at 100,000 cells/well on 96 wellPoly-D-Lysine coated plates. Irradiated K562-CD19 target cells wereadded at an effector to target ratio of 2.5:1. Cells were cultured forup to 4 days in the absence of inhibitor, or in the presence ibrutinib(concentrations of 5000, 500, 50, 5 and 0.5 nM) or in the presence ofthe compound of Formula (II) (concentrations 5000, 1582, 500, 158 and 50nM), for the duration of the culture. Cells were harvested at 1, 2, 3,and 4 days, and were analyzed by flow cytometry for T cell activationand differentiation surface markers CD69, CD107a, PD-1, CD25, CD38,CD39, CD95, CD62L, CCR7, CD45RO and for EGFRt (a surrogate marker forCAR-transduced cells).

Across the 3 different anti-CD19 CAR T cell donors, ibrutinib atconcentrations of 5000, 500, 50, 5 and 0.5 nM had no significant effecton expression of the EGFRt surrogate marker, or on any of the activationmarkers CD25, CD38, CD39, CD95 or CD62L, or on any of the T cellphenotypic markers assessed in this study (CCR7, CD62L and CD45RO),consistent with a conclusion that ibrutinib did not significantly impactthe activation state and/or differentiation subtype of the T cells inthis assay. Similar results were observed for culture in the presence ofthe compound of Formula (II). FIGS. 2A and 2B depict results forexemplary markers. The results in FIGS. 2C and 2D show that treatmentwith ibrutinib or the compound of Formula (II), respectively, did notaffect the phenotype of cells as central (TCM) or effector (TEM) memorysubsets as assessed by the expression of CCR7 and CD45RA. As shown inFIGS. 2E and 2F, there was a subtle decrease in expression levels ofCD69, CD107a or PD-1 when CD4+ or CD8+ cells, respectively, werecultured in the presence of ibrutinib. Similar results were observed forculture in the presence of the compound of Formula (II) (FIGS. 2G and2H). Some donor variability over time and magnitude was observed.

C. Cytokine Production

The production of cytokines by anti-CD19 CAR T cells cultured in thepresence or absence of ibrutinib or the compound of Formula (II) wereassessed by assessing cytokine levels in the supernatants of co-culturesof CAR-T cells and irradiated K562-CD19 target cells. CAR-T cellsgenerated as described above were plated at 100,000 cells/well on 96well Poly-D-Lysine coated plates. Irradiated target cells (K562-CD19)were added at an effector to target ratio of 2.5:1. Cells were culturedfor up to 4 days in the absence of inhibitor, in the presence of thecompound of Formula (II) at concentrations of 50, 150, 500, 1500 or 5000nM, or in the presence of ibrutinib at concentrations of 0.5, 5, 50 or500 nM. Culture supernatants were harvested at day 2, 3 or 4, and IFNγ,IL-2 and TNFα were measured from the culture supernatants using cytokinekits from Meso Scale Discovery (MSD). The secreted IFNγ, IL-2, and TNFαlevels from three healthy donor derived anti-CD19 CAR T cell lots overtime following co-culture with irradiated K562.CD19 target cells in thepresence of ibrutinib or the compound of Formula (II) is shown in FIG.3A and FIG. 3B, respectively. Absolute change in cytokine production, 2days after stimulation, of CAR-T cells generated from donor 2 in cellstreated with ibrutinib (FIG. 3C) or the compound of Formula (II) (FIG.3D), each in 2 independent experiments (mean±SEM), was determined. Asshown in FIGS. 3C and 3D, physiological concentrations of ibrutinib andthe compound of Formula (II), respectively, did not significantlyinhibit cytokine concentrations. Although the magnitude of responsesdiffered between donors, ibrutinib or the compound of Formula (II)overall did not inhibit CAR T cytokine production following stimulationwith antigen-specific K562.CD19 cells. The compound of Formula (II)modestly increased cytokine production in some donors, and similareffects were observed with 50 nM of ibrutinib. In contrast, a meandecrease in IL-2 of 19.6% or 1200 pg/mL was observed with 500 nMibrutinib (P<0.05) (FIG. 3C). Similar results were obtained when IL-4and IL-10 were measured.

D. Serial Restimulation

The ability of cells to expand ex vivo following repeated stimulationsin some aspects can indicate capacity of CAR-T cells to persist (e.g.,following initial activation) and/or is indicative of function and/orfitness in vivo (Zhao et al. (2015) Cancer Cell, 28:415-28). Anti-CD19CAR-T cells generated as described above were plated in triplicate at100,000 cells/well on 96 well Poly-D-Lysine coated plates, andirradiated target cells (K562-CD19) were added at an effector to targetratio of 2.5:1. Cells were stimulated in the presence of 500 and 50 nMibrutinib or 1581 and 158 nM of the compound of Formula (II), harvestedevery 3-4 days and counted, and cultured for restimulation with newtarget cells using the same culture conditions and added concentrationof ibrutinib or the compound of Formula (II) after resetting cell numberto initial seeding density for each round. A total of 7 rounds ofstimulation during a 25 day culture period were carried out.

For each round of stimulation, the fold change in cell number and thenumber of doublings was determined. The results for population doublingsare shown in FIGS. 4A and 4B, for ibrutinib and the compound of Formula(II), respectively. In this study, neither inhibitor was observed toimpact the initial growth of the CAR+ T cells. Expansion kinetics weresimilar in all treatment groups after three rounds of stimulation (day11), as observed by fold change and number of population doublings. Byday 18, however, for CAR+ T cells generated from two of the three donorsin this study, expansion (as measured by population doublings),following multiple rounds of restimulation, was observed to be enhancedby the presence of ibrutinib or the compound of Formula (II), at each ofthe concentrations assessed for the respective inhibitors as indicatedby enhanced cell numbers and increased numbers of population doublings.The cells derived from the two donors in which these differences wereobserved, generally, as compared to those derived from the other donor,performed less well in the serial restimulation assay in the absence ofeither inhibitor.

FIGS. 4C and 4D summarize the results of the number of cells in cultureat day 18 (5 rounds of restimulation) after stimulation for the threedonors in the presence of ibrutinib or the compound of Formula (II),respectively. As shown, a statistically significant increase in cellnumber after 18 days of the serial stimulation assay was observed. Inparticular, after five rounds of stimulation (day 18), CAR T cells fromdonor 2 treated with either ibrutinib or the compound of Formula (II) atthe highest concentrations had significantly (P<0.05) increased cellcounts relative to control cells. Non-significant, increased cell countswere also observed for cells from donor 3 treated with ibrutinib or thecompound of Formula (II) at the highest concentration tested. Whenassessing cell counts across control conditions, cells derived fromdonors 2 and 3 exhibited inferior performance to donor 1 cells in thisassay. Also, the cells derived from the two donors in which thesedifferences were observed, generally, as compared to those derived fromthe other donor, performed less well in the serial restimulation assayin the absence of either inhibitor. Notably, these donors with inferiorperformance benefited from treatment with ibrutinib or the compound ofFormula (II) in this assay. The results were consistent with the utilityof ibrutinib or the compound of Formula (II) or other Btk (or other TECfamily kinase) inhibitor, including selective inhibitors that do notinhibit Itk, to improve function, survival and/or expansion of T cellsimpaired in one or more factors impacting T cell function, survivaland/or proliferative capacity. For example, such combinations mayimprove T cell function and/or persistence following antigen encounter,even in the case of kinase inhibitors not capable of specificallyinhibiting Itk.

E. TH1 Phenotype

An assay was carried out to assess the skewing of anti-CD19 CART cellstowards a TH1 phenotype when cultured in the presence of ibrutinib orthe compound of Formula (II). Ibrutinib has been observed to limit Th2CD4 T cell activation and proliferation through the inhibition of ITK(Honda, F., et al. (2012) Nat Immunol, 13(4): 369-78). A serialrestimulation assay was performed as described above and cells wereharvested at various times and analyzed by flow cytometry to assesspercentage of TH1-phenotype (assessed as CD4+CXCR3+CRTH2−) T cells orTH2-phenotype (assessed as CD4+CXCR3−CRTH2+). Representative plots forcells cultured with and without indicated concentrations of ibrutinib orthe compound of Formula (II) is shown in FIG. 5A, and percentage of TH1cells following culture over the course of the serial restimulation, andunder various concentrations of ibrutinib or the compound of Formula(II), is shown in FIGS. 5B and 5C, respectively.

The presence of ibrutinib, but not the compound of Formula (II), wasobserved in this assay to increase the percentage of CAR+ T cellsobserved to exhibit a TH1 phenotype, after serial stimulation, and theeffect was observed to be greater with increasing concentrations ofibrutinib. During the 18-day serial stimulation period, the percentageof CAR T TH1 cells increased for all three donors under controlconditions (FIG. 5B). 500 nM ibrutinib further enhanced the percentageof TH1 cells (P<0.01), whereas no significant effects were observed withthe compound of Formula (II) treatment (FIG. C). The compound of Formula(II) has been reported to have >200 times lower affinity for ITKcompared with ibrutinib (Byrd, J. C., et al. (2016) N Engl J Med,374(4): 323-32), indicating that ibrutinib-mediated TH1 skewing could beinfluenced by off-target ITK activity.

No significant effects of either inhibitor on additional CAR Tactivation or memory markers were observed in CAR T cells isolated fromthe serial stimulation assay (FIGS. 5D and 5E, compound of Formula (II);and FIGS. 5F and 5G, ibrutinib).

Example 2: Enhancement of Anti-Tumor Activity of CAR-Expressing T Cellsin the Presence of an Inhibitor of a TEC Family Kinase

A disseminated tumor xenograft mouse model was generated by injectingNOD/Scid/gc−/−(NSG) mice with cells of a CD19+ Nalm-6 disseminated tumorline, identified to be resistant to BTK inhibition.

On day zero (0), NSG mice were intravenously injected with 5×10⁵ Nalm-6cells expressing firefly luciferase. Beginning at day 4 and daily forthe duration of the study, mice were treated with a vehicle control orwere treated with ibrutinib, in each case by daily oral gavage (P.O.) at25 m/kg q.d. To permit assessment of the effect of a combination therapywith the inhibitor, a suboptimal dose of anti-CD19 CART cells from twodifferent donors were i.v. injected into the mice at 5×10⁵/mouse at day5. As a control, mice were administered the vehicle control or ibrutinibbut without administration of the CAR-T cells. Eight (N=8) mice pergroup were monitored.

Following treatment as described above, tumor growth over time wasmeasured by bioluminescence imaging and the average radiance(p/s/cm²/sr) was measured. Results are shown in FIG. 6A for tumor growthover time from mice treated with ibrutinib and CAR T cells. Analysis ofthe results from the same study monitoring tumor growth at greater timepoints post-tumor injection from two different donors is shown in FIG.6B. As shown, ibrutinib treatment alone had no effect on tumor burden inthis ibrutinib-resistant model compared to vehicle treatment. Incontrast, mice administered CAR-T cells and ibrutinib exhibited asignificantly decreased tumor growth compared to mice treated with CAR-Tcells and vehicle control (p<0.001, ***).

The combined administration of CAR T and ibrutinib also was observed toresult in significantly increased survival compared with the CAR T andvehicle condition, as shown by Kaplan Meier curves showing survival oftumor bearing mice treated with ibrutinib and CAR T cells. As shown inFIG. 6C, mice treated with CAR-T cells and ibrutinib exhibited anincreased median survival compared to the group receiving the suboptimalanti-CD19 CAR T cell dose+vehicle. Analysis of the results from the samestudy monitoring survival at greater time points post-tumor injectionfrom two different donors is shown in FIG. 6D, which showed that thecombined administration of CAR T and ibrutinib also was observed toresult in significantly increased survival compared with the CAR T andvehicle condition, (p<0.001, ***).

Example 3: Assessment of CAR-Expressing T Cell Phenotype, Function andAnti-Tumor Activity In Vivo in the Presence of an Inhibitor of a TECFamily Kinase

NSG mice described in Example 2 were intravenously injected on day 0with 5×10⁵ Nalm-6 cells expressing firefly luciferase. Beginning at day4 and daily for the duration of the study, mice were treated with avehicle control or were treated daily with an inhibitor of a TEC familykinase, either ibrutinib or the compound of Formula (II), in drinkingwater (D.W.) at 25 mg/kg/day. A bridging experiment confirmed thatadministration of ibrutinib by drinking water was equivalent to oralgavage administration (data not shown). To permit assessment of theeffect of a combination therapy with the inhibitor, a suboptimal dose ofanti-CD19 CAR T cells was i.v. injected into the mice at 5×10⁵/mouse atday 5. As a control, mice were administered the vehicle control withoutadministration of the CAR-T cells or inhibitor.

Following treatment as described above, the tumor growth and percentsurvival of treated mice was determined. As shown in FIG. 7A, micetreated with anti-CD19 CAR-T cells and either ibrutinib or the compoundof Formula (II) exhibited an increased median survival compared to thegroup receiving the suboptimal anti-CD19 CAR T cell dose+vehicle(p<0.001). The compound of Formula (II) or ibrutinib, administered incombination with CAR T, also significantly (P<0.001) decreased tumorgrowth (FIG. 7B) compared with the CAR T administered with vehiclealone. The results were similar using anti-CD19 CAR-T cells generated byengineering T cells derived from two different donors.

Pharmacokinetic analysis of CAR+ T cells was analyzed in blood and bonemarrow from mice having received anti-CD19 CAR+ T cells from onedonor-derived cells, and that had been treated with vehicle, ibrutinibor the compound of Formula (II) (3 mice per group), Samples wereanalyzed to assess presence and levels of EGFRt+ CAR T cells and/ortumor cells at day 7, 12, 19 and 26 post CAR+ T cell transfer. As shownin FIGS. 7C and 7E, a significant increase in circulating CAR+ T cellswas observed in mice treated with ibrutinib and the compound of Formula(II), respectively, as compared to those treated with CAR+ T cells andvehicle, consistent with a greater expansion of CAR-T cells in the bloodin the presence of the inhibitor. At day 19 post CAR-T cell transfer, asignificant increase in the number of cells in the blood was observedafter treatment with ibrutinib (FIG. 7D; *p<0.05) and the compound ofFormula (II) (FIG. 7E; ***p<0.001), respectively. As shown in FIGS. 7Gand 7H, significantly fewer tumor cells were detected in the blood orbone marrow in mice in which the CAR+ cell treatment was combined withtreatment with ibrutinib or the compound of Formula (II), respectively,as compared to with vehicle alone.

Ex vivo immunophenotyping also was performed on CAR+ T cells harvestedfrom bone marrow at day 12 post-CAR T from mice that received CAR+ Tcells and that had been treated with vehicle, ibrutinib or the compoundof Formula (II) (n=3 mice per group). Cells were assessed for surfacemarkers CD44, CD45RA, CD62L, CD154, CXCR3, CXCR4, PD-1 by flow cytometryand T-distributed stochastic neighbor embedding (t-SNE) high dimensionalanalysis was performed using FlowJo software. As shown in FIG. 8A,changes were observed in CAR+ T cells isolated from the bone marrow ofanimals having received CAR-T cells in combination with ibrutinib or thecompound of Formula (II), as compared to with vehicle alone (control).Using multivariate t-SNE FACS analysis based on pooled analysis fromthree mice per group, 4 distinct population clusters were identified(FIG. 8B). FACs histograms showing the individual expression profiles ofCD4, CD8, CD62L, CD45RA, CD44 and CXCR3 from the 4 gated t-SNE in FIG.8A overlaid on the expression of the total population (shaded) is shownin FIG. 8C. The percentage and fold change of each t-SNE population incontrol mice or mice treated with ibrutinib or the compound of Formula(II), is shown in FIG. 8D. Statistically significant differences areindicated as P<0.95 (*), P<0.01 (**), P<0.001 (***), P<0.0001 (****).

An increase in CD8+CD44^(hi) CXCR3^(hi) CD45RA^(lo) CD62L^(hi)(population 2) and CD4+CD44^(hi) CXCR3^(int) CD45RA^(hi) CD62L(population 4) was observed in the bone marrow of CAR T-treated micealso administered ibrutinib or the compound of Formula (II) as comparedto control mice, at day 12 post CAR T transfer (FIG. 8B-8C). Micetreated with the compound of Formula (II) exhibited a greater enrichmentof population 2 as compared to that observed in ibrutinib treatedanimals (populations 2 and 4 representing 29.2% and 8.4% of CAR-T cellsin these mice, respectively), whereas a greater enhancement ofpopulation 4 was observed in ibrutinib-treated animals (15.2% comparedto 4.4% of CAR-T cells) (FIG. 8D).

Example 4: Enhancement of Cytolytic Function of CAR-Expressing T CellsManufactured from Diffuse Large B-Cell Lymphoma (DLBCL) Patients in thePresence of an Inhibitor of a TEC Family Kinase

Anti-CD19 CAR-T cells were generated substantially as described inExample 1, except that T cells were isolated from two exemplary patientshaving diffuse large B-cell lymphoma (DLBCL). Cells were subjected toserial restimulation as described in Example 1.D, by co-culturing CAR+ Tcells with K562-CD19 targets cells at an effector to target ratio of2.5:1 and in the presence of 500 and 50 nM ibrutinib, harvesting cellsevery 3-4 days and restimulating under the same conditions afterresetting cell number. Cells were subjected to serial restimulation overa 21 day culture period and monitored for cell expansion and cytotoxicactivity.

As shown in FIG. 9A, cell expansion, as determined by the number of celldoublings, was observed during the 21 day culture period from cellsderived from each individual subject. Ibrutinib did not inhibit theproliferation of CAR T cells derived from either patient (FIG. 9A), anobservation consistent with previous data from healthy donor-derived CART cells. CAR+ T cells manufactured from cells derived from eachindividual subject demonstrated an increase in cytolytic function in thepresence of 500 nM ibrutinib after 16 days of serial restimulation (P<0.001) (FIG. 9B). In cells derived from one patient, an increase incytolytic activity after 16 days of serial stimulation was observed with50 nM ibrutinib (P<0.01) (FIG. 9B). This increase in cytolytic activityis consistent with results from healthy donor cells (FIG. 1C, D).

Example 5: Assessment of Molecular Signature by RNA-Seq ofCAR-Expressing T Cells Treated with Ibrutinib

RNA was isolated from individual CAR-expressing cells, derived fromthree different donors, that had been treated for 18 days in a serialstimulation assay in the presence of ibrutinib (50 nM, 500 nM), thecompound of formula (II) (1581 nM) or control (0 nM). RNA isolation wasperformed using the RNEasy Micro Kit (Qiagen). Samples were sequencedand RNASeq reads were mapped to the human genome (GRCh38) and aligned tothe GENCODE release 24 gene model. RNAseq quality metrics were generatedand evaluated to confirm consistency across samples. Differentiallyexpressed genes were identified by imposing a log₂ fold change cutoff of0.5 and a Benjamini-Hochberg adjusted false discovery rate (FDR) cutoffof 0.05.

As shown in the volcano plot in FIG. 10A, 500 nM ibrutinib significantly(FDR<0.05, absLog2FC>0.5) altered the expression of 41 protein-codinggenes. FIG. 10C shows a heat map of gene expression changes for thegenes identified in FIG. 10A. In a separate experiment under similarculture conditions, only 3 genes were significantly altered (FDR<0.05,absLog₂FC>0.5) under treatment with the compound of formula (II) (1581nM). FIG. 10B depicts a Volcano plot of expressed genes from day 18serially stimulated CAR T cells treated with 1581 nM of the compound ofFormula (II) compared with control.

Box plots of gene expression for two exemplary genes following treatmentwith different concentrations of inhibitor (50 nM or 500 nM) or controlare shown in FIGS. 11A-B. Decreases in genes such as granzyme A (FIG.11A) and increases in SELL/CD62L (FIG. 11B) are consistent with aneffect of ibrutinib to dampen terminal-effector-like genes whileenhancing genes associated with memory development. Furthermore, RNA-Seqrevealed that genes associated with promoting TH1 differentiation werealtered by ibrutinib, including upregulation of MSC, known to suppressTH2 programing (Wu, C., et al. (2017) Nat Immunol, 18(3): 344-353), anddownregulation of HES6, HIC1, LZTFL1, NRIP1, CD38 and RARRES3,associated with the ATRA/Retinoic acid signaling pathway identified toinhibit TH1 development (Britschgi, C., et al. (2008) Br J Haematol,141(2): 179-87; Jiang, H., et al. (2016) J Immunol, 196(3): 1081-90;Heim, K. C., et al. (2007) Mol Cancer, 6: 57; Nijhof, I. S., et al.(2015) Leukemia, 29(10): 2039-49; Zirn, B., et al. (2005) Oncogene,24(33): 5246-51) (FIG. 11E-B-D). In support of the RNA-Seq results, asignificant increase in CD62L expression was observed by flow cytometryafter 18 days of serial stimulation in donors 2 and 3 (FIGS. 12A and12B). Taken together, these results support that long term ibrutinibtreatment may result in an increased TH1 and memory-like phenotype inCAR T.

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

SEQUENCES SEQ ID NO. SEQUENCE DESCRIPTION  1 ESKYGPPCPPCP spacer(IgG4hinge) (aa) Homo sapiens  2 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTspacer (IgG4hinge) (nt) Homo sapiens  3ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI Hinge-CH3 spacerAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCS Homo sapiensVMHEALHNHYTQKSLSLSLGK  4ESKYGPPCPPCPAPEFLGGPSVELFPPKPKDTLMISRTPEVTCVVVDVS Hinge-CH2-CH3QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWING spacerKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL Homo sapiensTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK  5RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKE IgD-hinge-FcKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSD Homo sapiensLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH  6 LEGGGEGRGSLLTCGDVEENPGPRT2A artificial  7 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTtEGFR HTPPLDPQELDILKTVKEITGELLIQAWPENRTDLHAFENLEIIRGRTK artificialQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLEM  8 FWVLVVVGGVLACYSLLVTVAFIIFWVCD28 (amino acids 153-179 of Accession No. P10747) Homo sapiens  9IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP CD28 (aminoFWVLVVVGGVLACYSLLVTVAFIIFWV acids 114-179 of Accession No. P10747)Homo sapiens 10 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (aminoacids 180-220 of P10747) Homo sapiens 11RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (LL to GG) Homo sapiens12 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (aminoacids 214-255 of Q07011.1) Homo sapiens 13RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3 zetaRRKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY Homo sapiensQGLSTATKDTYDALHMQALP PR 14RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3 zetaRRKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY Homo sapiensQGLSTATKDTYDALHMQALP PR 15RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3 zetaRRKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY Homo sapiensQGLSTATKDTYDALHMQALP PR 16 PGGG-(SGGGG)5-P- wherein P is proline, G islinker glycine and S is serine 17 GSADDAKKDAAKKDGKS linker 18MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRGSKKG Tyrosine-proteinSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDE kinase BTKGPLYVESPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNA Homo sapiensMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDEGLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILL SNILDVMDEES 19AACTGAGTGGCTGTGAAAGGGTGGGGTTTGCTCAGACTGTCCTTCCTCTCTGGA Tyrosine-proteinCTGTAAGAATATGTCTCCAGGGCCAGTGTCTGCTGCGATCGAGTCCCACCTTCC kinase BTKAAGTCCTGGCATCTCAATGCATCTGGGAAGCTACCTGCATTAAGTCAGGACTGA Homo sapiensGCACACAGGTGAACTCCAGAAAGAAGAAGCTATGGCCGCAGTGATTCTGGAGAGCATCTTTCTGAAGCGATCCCAACAGAAAAAGAAAACATCACCTCTAAACTTCAAGAAGCGCCTGTTTCTCTTGACCGTGCACAAACTCTCCTACTATGAGTATGACTTTGAACGTGGGAGAAGAGGCAGTAAGAAGGGTTCAATAGATGTTGAGAAGATCACTTGTGTTGAAACAGTGGTTCCTGAAAAAAATCCTCCTCCAGAAAGACAGATTCCGAGAAGAGGTGAAGAGTCCAGTGAAATGGAGCAAATTTCAATCATTGAAAGGTTCCCTTATCCCTTCCAGGTTGTATATGATGAAGGGCCTCTCTACGTCTTCTCCCCAACTGAAGAACTAAGGAAGCGGTGGATTCACCAGCTCAAAAACGTAATCCGGTACAACAGTGATCTGGTTCAGAAATATCACCCTTGCTTCTGGATCGATGGGCAGTATCTCTGCTGCTCTCAGACAGCCAAAAATGCTATGGGCTGCCAAATTTTGGAGAACAGGAATGGAAGCTTAAAACCTGGGAGTTCTCACCGGAAGACAAAAAAGCCTCTTCCCCCAACGCCTGAGGAGGACCAGATCTTGAAAAAGCCACTACCGCCTGAGCCAGCAGCAGCACCAGTCTCCACAAGTGAGCTGAAAAAGGTTGTGGCCCTTTATGATTACATGCCAATGAATGCAAATGATCTACAGCTGCGGAAGGGTGATGAATATTTTATCTTGGAGGAAAGCAACTTACCATGGTGGAGAGCACGAGATAAAAATGGGCAGGAAGGCTACATTCCTAGTAACTATGTCACTGAAGCAGAAGACTCCATAGAAATGTATGAGTGGTATTCCAAACACATGACTCGGAGTCAGGCTGAGCAACTGCTAAAGCAAGAGGGGAAAGAAGGAGGTTTCATTGTCAGAGACTCCAGCAAAGCTGGCAAATATACAGTGTCTGTGTTTGCTAAATCCACAGGGGACCCTCAAGGGGTGATACGTCATTATGTTGTGTGTTCCACACCTCAGAGCCAGTATTACCTGGCTGAGAAGCACCTTTTCAGCACCATCCCTGAGCTCATTAACTACCATCAGCACAACTCTGCAGGACTCATATCCAGGCTCAAATATCCAGTGTCTCAACAAAACAAGAATGCACCTTCCACTGCAGGCCTGGGATACGGATCATGGGAAATTGATCCAAAGGACCTGACCTTCTTGAAGGAGCTGGGGACTGGACAATTTGGGGTAGTGAAGTATGGGAAATGGAGAGGCCAGTACGACGTGGCCATCAAGATGATCAAAGAAGGCTCCATGTCTGAAGATGAATTCATTGAAGAAGCCAAAGTCATGATGAATCTTTCCCATGAGAAGCTGGTGCAGTTGTATGGCGTCTGCACCAAGCAGCGCCCCATCTTCATCATCACTGAGTACATGGCCAATGGCTGCCTCCTGAACTACCTGAGGGAGATGCGCCACCGCTTCCAGACTCAGCAGCTGCTAGAGATGTGCAAGGATGTCTGTGAAGCCATGGAATACCTGGAGTCAAAGCAGTTCCTTCACCGAGACCTGGCAGCTCGAAACTGTTTGGTAAACGATCAAGGAGTTGTTAAAGTATCTGATTTCGGCCTGTCCAGGTATGTCCTGGATGATGAATACACAAGCTCAGTAGGCTCCAAATTTCCAGTCCGGTGGTCCCCACCGGAAGTCCTGATGTATAGCAAGTTCAGCAGCAAATCTGACATTTGGGCTTTTGGGGTTTTGATGTGGGAAATTTACTCCCTGGGGAAGATGCCATATGAGAGATTTACTAACAGTGAGACTGCTGAACACATTGCCCAAGGCCTACGTCTCTACAGGCCTCATCTGGCTTCAGAGAAGGTATATACCATCATGTACAGTTGCTGGCATGAGAAAGCAGATGAGCGTCCCACTTTCAAAATTCTTCTGAGCAATATTCTAGATGTCATGGATGAAGAATCCTGAGCTCGCCAATAAGCTTCTTGGTTCTACTTCTCTTCTCCACAAGCCCCAATTTCACTTTCTCAGAGGAAATCCCAAGCTTAGGAGCCCTGGAGCCTTTGTGCTCCCACTCAATACAAAAAGGCCCCTCTCTACATCTGGGAATGCACCTCTTCTTTGATTCCCTGGGATAGTGGCTTCTGAGCAAAGGCCAAGAAATTATTGTGCCTGAAATTTCCCGAGAGAATTAAGACAGACTGAATTTGCGATGAAAATATTTTTTAGGAGGGAGGATGTAAATAGCCGCACAAAGGGGTCCAACAGCTCTTTGAGTAGGCATTTGGTAGAGCTTGGGGGTGTGTGTGTGGGGGTGGACCGAATTTGGCAAGAATGAAATGGTGTCATAAAGATGGGAGGGGAGGGTGTTTTGATAAAATAAAATTACTAGAAAGCTTGAAAGTC

1.-123. (canceled)
 124. A method of treatment, the method comprising:(a) administering, to a subject having a disease or condition, T cellsthat specifically recognize or specifically bind to an antigenassociated with, or expressed or present on cells of, the disease orcondition; and (b) administering to the subject an inhibitor of a targetprotein tyrosine kinase, wherein the inhibitor does not inhibitinterleukin-2-inducible kinase (ITK) and/or inhibits ITK with ahalf-maximal inhibitory concentration (IC₅₀) of greater than or greaterthan about 1000 nM.
 125. The method of claim 124, wherein the targetprotein tyrosine kinase is a tyrosine kinase expressed in hepatocellularcarcinoma (TEC) family kinase.
 126. The method of claim 125, wherein thetarget protein tyrosine kinase is a tyrosine kinase expressed inhepatocellular carcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), aBMX non-receptor tyrosine kinase (BMX/ETK), or a receptortyrosine-protein kinase ErbB4 (ERBB4).
 127. The method of claim 124,wherein the inhibitor is selected from the group consisting of thecompound of Formula (II)

ONO/GS-4059, Compound 30 or Compound 38, GDC-0834; RN-486; CGI-560;CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.
 128. Themethod of claim 124, wherein the inhibitor comprises the compound ofFormula (II):

or an enantiomer, a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof or a pharmaceutical compositioncomprising any of the foregoing.
 129. A method of treatment, the methodcomprising: (1) administering, to a subject having a disease orcondition, T cells that specifically recognize or specifically bind toan antigen associated with the disease or condition; and (2)administering to the subject an inhibitor of a target protein tyrosinekinase, which target protein tyrosine kinase is a tyrosine kinaseexpressed in hepatocellular carcinoma (TEC), a resting lymphocyte kinase(RLK/TXK), a BMX non-receptor tyrosine kinase (BMX/ETK), or a receptortyrosine-protein kinase ErbB4 (ERBB4), wherein: the inhibitor is aselective inhibitor of the target protein tyrosine kinase; and/or theinhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC₅₀) that is at least 10 or atleast 100 times lower than that of the IC₅₀ of the inhibitor for anyprotein tyrosine kinase or TEC family kinase distinct from the targetprotein tyrosine kinase, and/or inhibits the target protein tyrosinekinase with an IC₅₀ at least 2, at least 10 or at least 100 times lowerthan that the IC₅₀ value of the inhibitor for bothinterleukin-2-inducible kinase (ITK) and Bruton's tyrosine kinase (BTK);and/or the inhibitor inhibits the target protein tyrosine kinase with ahalf-maximal inhibitory concentration (IC₅₀) of less than or less thanabout 1000 nM or less.
 130. The method of claim 129, wherein theinhibitor is not ibrutinib.
 131. The method of claim 129, wherein theinhibitor does not inhibit ITK and/or inhibits ITK with a half-maximalinhibitory concentration (IC₅₀) of greater than or greater than about1000 nM.
 132. The method of claim 129, wherein the inhibitor comprisesis a compound of Formula (II):

or an enantiomer, a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof or a pharmaceutical compositioncomprising any of the foregoing.
 133. The method of claim 124, whereinthe disease or condition (i) is not a B cell-derived disease orcondition (ii) is not associated with expression of CD19, CD22, or CD20;(iii) does not express the target protein tyrosine kinase, (iv) does notcontain a form of the target protein tyrosine kinase that is sensitiveto the inhibitor, (v) does not contain a kinase sensitive to theinhibitor and/or (vi) is not sensitive to inhibition by the inhibitorand/or wherein the subject or disease or condition is resistant orrefractory to the inhibitor and/or to an inhibitor of BTK and/or thetarget kinase is not ordinarily expressed or is not suspected of beingexpressed in cells from which the disease or condition is derived. 134.The method of claim 124, wherein: (i) the subject and/or the disease orcondition (a) is resistant to inhibition of Bruton's tyrosine kinase(BTK) and/or (b) comprises a population of cells that are resistant toinhibition by the inhibitor; (ii) the subject and/or the disease orcondition comprises a mutation or disruption in a nucleic acid encodingBTK, capable of reducing or preventing inhibition of the BTK by theinhibitor and/or by ibrutinib; and/or (iii) at the time of theadministration in (1) and at the time of the administration in (2) thesubject has relapsed following remission after treatment with, or beendeemed refractory to treatment with the inhibitor and/or with a BTKinhibitor therapy.
 135. The method of claim 134, wherein the mutation inthe nucleic acid encoding BTK comprises a C481S or C481R substitution,and/or a T474I or T474M substitution.
 136. The method of claim 124,wherein: the target protein tyrosine kinase is not expressed by cells ofthe disease or condition, is not ordinarily expressed or is notsuspected of being expressed in cells from which the disease orcondition is derived, and/or the disease or condition is not sensitiveto the inhibitor; and/or at least a plurality of the T cells express thetarget protein tyrosine kinase; and/or the target protein tyrosinekinase is expressed in T cells.
 137. The method of claim 124, whereinthe disease or condition is a cancer.
 138. The method of claim 137,wherein the disease or condition is a cancer selected from the groupconsisting of sarcomas, carcinomas, lymphomas, non-Hodgkin lymphomas(NHLs), diffuse large B cell lymphoma (DLBCL), leukemia, CLL, ALL, AMLand myeloma.
 139. The method of claim 124, wherein the T cells recognizeor target an antigen selected from ROR1, B cell maturation antigen(BCMA), tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, andhepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30,CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbBdimers, EGFR vIII, FBP, FCRLS, FCRHS, fetal acethycholine e receptor,GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain,Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associatedantigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen ofmelanoma (PRAIVIE), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptora2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1,HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6integrin, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands,CD44v6, dual antigen, and an antigen associated with a universal tag, acancer-testes antigen, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), prostate specific antigen, PSMA,Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, and a pathogen-specific antigen. 140.The method of claim 124, wherein the T cells comprise tumor infiltratinglymphocytes (TILs) or comprises genetically engineered T cellsexpressing a recombinant receptor that specifically binds to an antigen.141. The method of claim 140, wherein the recombinant receptor is atransgenic T cell receptor (TCR) or a chimeric antigen receptor (CAR).142. The method of claim 141, wherein the chimeric antigen receptor(CAR) comprises an extracellular antigen-recognition domain thatspecifically binds to the antigen and an intracellular signaling domaincomprising an ITAM.
 143. The method of claim 142, wherein theintracellular signaling domain comprises an intracellular domain of aCD3-zeta (CD3) chain.
 144. The method of claim 142, wherein the chimericantigen receptor (CAR) further comprises a costimulatory signalingregion.
 145. The method of claim 144, wherein the costimulatorysignaling region comprises a signaling domain of CD28 or 4-1BB.
 146. Themethod of claim 124, wherein the inhibitor irreversibly reduces oreliminates the activation of the target protein tyrosine kinase,specifically binds to a binding site in the active site of the targetprotein tyrosine kinase comprising an amino acid residue correspondingto residue C481 in the sequence set forth in SEQ ID NO:18, and/orreduces or eliminates autophosphorylation activity of the target proteintyrosine kinase.
 147. The method of claim 124, wherein the inhibitor isadministered concurrently with or subsequently to initiation ofadministration of the T cells.
 148. The method of claim 124, wherein theinhibitor is administered subsequently to initiation of administrationof the T cells.
 149. The method of claim 148, wherein the inhibitor isadministered within, or within about, 1 hour, 2 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours or 1 week of theinitiation of the administration of the T cells.
 150. The method ofclaim 148, wherein the inhibitor is administered at a time in which: thenumber of administered T cells detectable in the blood from the subjectis decreased compared to in the subject at a preceding time point afterinitiation of the administration of the T cells; the number ofadministered T cells detectable in the blood is less than or less thanabout 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold or100-fold or less the peak or maximum number of the cells of the T celltherapy detectable in the blood of the subject after initiation ofadministration of the administration of the T cells; and/or at a timeafter a peak or maximum level of the administered T cells are detectablein the blood of the subject, the number of cells of or derived from theT cells detectable in the blood from the subject is less than less than10%, less than 5%, less than 1% or less than 0.1% of total peripheralblood mononuclear cells (PBMCs) in the blood of the subject.
 151. Themethod of claim 124, wherein the administration of the inhibitor iscontinued, from at least after initiation of administration of the Tcells, until: the number of cells of or derived from the T cellsadministered detectable in the blood from the subject is increasedcompared to in the subject at a preceding time point just prior toadministration of the inhibitor or compared to a preceding time pointafter administration of the T-cell therapy; the number of cells of orderived from the T cells detectable in the blood is within 2.0-fold(greater than or less than) the peak or maximum number observed in theblood of the subject after initiation of administration of the T cells;the number of cells of the T cells detectable in the blood from thesubject is greater than or greater than about 10%, 15%, 20%, 30%, 40%,50%, or 60% total peripheral blood mononuclear cells (PBMCs) in theblood of the subject; and/or the subject exhibits a reduction in tumorburden as compared to tumor burden at a time immediately prior to theadministration of the T cells or at a time immediately prior to theadministration of the inhibitor; and/or the subject exhibits complete orclinical remission.
 152. The method of claim 124, wherein the inhibitoris administered orally.
 153. The method of claim 124, wherein theinhibitor is administered at a total daily dosage amount of at least orat least about 50 mg/day.
 154. The method of claim 124, wherein theinhibitor is administered in an amount that is at or about or less thanor at or about 200 mg/day.
 155. The method of claim 124, wherein theadministered T cells comprise CD4+ T cells and/or CD8+ T cells.
 156. Themethod of claim 124, wherein the administered T cells compriseadministration of a dose comprising a number of cells between at orabout 5×10⁵ cells/kg body weight of the subject and at or about 1×10⁷cells/kg body weight of the subject.
 157. The method of claim 124,wherein the administered T cells comprise administration of a dosecomprising a number of cells between at or about 1×10⁷ and at or about2×10⁸ total T cells comprising the recombinant receptor.
 158. The methodof claim 124, wherein the method further comprises administering alymphodepleting chemotherapy prior to administration of the T cellsand/or wherein the subject has received a lymphodepleting chemotherapyprior to administration of the T cells.
 159. The method of claim 124,wherein the method further comprises administering an immune modulatoryagent to the subject, wherein the administration of the cells and theadministration of the immune modulatory agent are carried outsimultaneously, separately or in a single composition, or sequentially,in either order.
 160. The method of claim 124, wherein the administeredT cells exhibit increased or prolonged expansion and/or persistence inthe subject as compared to a method in which the administered T cellsare administered to the subject in the absence of the inhibitor. 161.The method of claim 124, wherein the method reduces tumor burden to agreater degree and/or for a greater period of time as compared to thereduction that would be observed with a comparable method in which theadministered T cells are administered to the subject in the absence ofthe inhibitor.
 162. A combination, comprising: T cells expressing arecombinant receptor that binds to an antigen associated with a diseaseor condition, and an inhibitor of a target protein tyrosine kinase,wherein the inhibitor does not inhibit interleukin-2-inducible kinase(ITK) and/or inhibits ITK with a half-maximal inhibitory concentration(IC₅₀) of greater than or greater than about 1000 nM and/or the targetprotein tyrosine kinase is a tyrosine kinase expressed in hepatocellularcarcinoma (TEC), a resting lymphocyte kinase (RLK/TXK), a BMXnon-receptor tyrosine kinase (BMX/ETK), or a receptor tyrosine-proteinkinase ErbB4 (ERBB4).
 163. A combination, comprising: T cells comprisinga recombinant antigen receptor that specifically binds to an antigenassociated with a disease or condition; and a kinase inhibitor or apharmaceutical composition comprising the inhibitor, wherein theinhibitor comprises the compound of Formula (II):

or an enantiomer, pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof.
 164. A kit, comprising thecombination of claim 162 and instructions for administering, to asubject for treating a disease or condition, the genetically engineeredcells and the inhibitor.
 165. A kit, comprising the combination of claim163 and instructions for administering, to a subject for treating adisease or condition, the genetically engineered cells and theinhibitor.
 166. A method of engineering immune cells expressing arecombinant receptor, comprising: contacting a population of cellscomprising T cells with an inhibitor of a target protein tyrosinekinase, wherein the inhibitor does not inhibit interleukin-2-induciblekinase (ITK) and/or inhibits ITK with a half-maximal inhibitoryconcentration (IC₅₀) of greater than or greater than about 1000 nMand/or the target protein tyrosine kinase is a tyrosine kinase expressedin hepatocellular carcinoma (TEC), a resting lymphocyte kinase(RLK/TXK), a BMX non-receptor tyrosine kinase (BMX/ETK), or a receptortyrosine-protein kinase ErbB4 (ERBB4); and introducing a nucleic acidencoding a recombinant receptor into the population of T cells underconditions such that the recombinant receptor is expressed.
 167. Themethod of claim 166, wherein the population of cells is or comprises Tcells.
 168. The method of claim 167, wherein the population of cellscomprises CD4+ T cells and/or CD8+ T cells.
 169. The method of claim166, wherein the inhibitor comprises the compound of Formula (II):

or an enantiomer, pharmaceutically acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof or a pharmaceutical compositioncomprising any of the foregoing.
 170. A method of producing geneticallyengineered T cells, comprising introducing a nucleic acid moleculeencoding a recombinant receptor into a primary T cell, wherein the Tcells is from a subject having been administered an inhibitor of atarget protein tyrosine kinase, wherein the inhibitor does not inhibitinterleukin-2-inducible kinase (ITK) and/or inhibits ITK with ahalf-maximal inhibitory concentration (IC₅₀) of greater than or greaterthan about 1000 nM and/or the target protein tyrosine kinase is atyrosine kinase expressed in hepatocellular carcinoma (TEC), a restinglymphocyte kinase (RLK/TXK), a BMX non-receptor tyrosine kinase(BMX/ETK), or a receptor tyrosine-protein kinase ErbB4 (ERBB4).
 171. Themethod of claim 170, wherein the inhibitor comprises the compound ofFormula (II)

or an enantiomer, pharmaceutically-acceptable salt, solvate, hydrate,co-crystal, polymorph or prodrug thereof or a pharmaceutical compositioncomprising any of the foregoing.
 172. The method of claim 170, whereinthe T cells comprise CD4+ T cells and/or CD8+ T cells.